Journal: Cell division

Article Title: Long non-coding RNA NEAT1 induced by BHLHE40 activates Wnt/β-catenin signaling and potentiates colorectal cancer progression.

doi: 10.1186/s13008-024-00129-7

Figure Lengend Snippet: Fig. 2 BHLHE40 mediates the transcription of NEAT1 in CRC cells. A Genes co-expressed with NEAT1 in CRC were obtained from UALCAN. B The intersection of genes co-expressed with NEAT1 in COAD and READ and the transcription factors targeting NEAT1 downloaded from hTFtarget. C The expression of these eight transcription factors in CRC was analyzed in the UALCAN. D The expression of BHLHE40 in both COAD and READ. E The binding fragment of BHLHE40 on the NEAT1 promoter with the highest score. F RT-qPCR detection of BHLHE40 expression in CRC tissues and adjacent tissues by RT-qPCR (n = 54). G Expression of NEAT1 in CRC cell lines and HCoEpiC cells by RT-qPCR. H Knockdown efficiency of BHLHE40 by RT-qPCR. I RT-qPCR detection of NEAT1 expression after knockdown of BHLHE40 in LoVo and HCT-15 cells. J Changes in luciferase activity after knockdown of BHLHE40 using luciferase activity assay. K BHLHE40 binding to the NEAT1 promoter in CRC cell using ChIP assay. Results were expressed as magnitude of relative expression (means ± SD) from three independent experiments. F Paired t-test; G one-way ANOVA; H–K two-way ANOVA. *p < 0.05

Article Snippet: Afterward, the membranes were sealed with non-fat milk at room temperature for 2 h. Primary antibodies to BHLHE40 (1:1000, 17895-1-AP, ProteinTech Group, Chicago, IL, USA), Wnt (1:500, 27935-1-AP, ProteinTech Group), β-catenin (1:2000, 17565-1-AP, ProteinTech), cyclin D1 (1:5000, 26939-1-AP, ProteinTech), c-myc (1:500, GTX103436, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), E-cadherin (1:500, GTX100443, GeneTex), N-cadherin (1:500, GTX127345, GeneTex), Vimentin (1:5000, GTX100619, GeneTex), and β-actin (1:500, D110001, Sangon) were applied overnight at 4 °C.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Knockdown, Luciferase, Activity Assay

Journal: Cell division

Article Title: Long non-coding RNA NEAT1 induced by BHLHE40 activates Wnt/β-catenin signaling and potentiates colorectal cancer progression.

doi: 10.1186/s13008-024-00129-7

Figure Lengend Snippet: Fig. 4 NEAT1 reverses the metastasis-suppressing and tumor-suppressing properties of sh-BHLHE40. HCT-15 cells after infection were injected subcutaneously into nude mice to observe tumor growth. A Tumor growth curve. B Immunohistochemical analysis of BHLHE40 and Ki67 expression in xenograft tumor tissues of nude mice. C HCT-15 cells after infection were injected into nude mice via the tail vein, and lung metastasis formation was observed using HE staining. D Western blot for BHLHE40 and EMT-related protein expression in metastatic tissues with lung infiltration. Results were expressed as magnitude of relative expression (means ± SD) from six nude mice in each group. Two-way ANOVA. *p < 0.05

Article Snippet: Afterward, the membranes were sealed with non-fat milk at room temperature for 2 h. Primary antibodies to BHLHE40 (1:1000, 17895-1-AP, ProteinTech Group, Chicago, IL, USA), Wnt (1:500, 27935-1-AP, ProteinTech Group), β-catenin (1:2000, 17565-1-AP, ProteinTech), cyclin D1 (1:5000, 26939-1-AP, ProteinTech), c-myc (1:500, GTX103436, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), E-cadherin (1:500, GTX100443, GeneTex), N-cadherin (1:500, GTX127345, GeneTex), Vimentin (1:5000, GTX100619, GeneTex), and β-actin (1:500, D110001, Sangon) were applied overnight at 4 °C.

Techniques: Infection, Injection, Immunohistochemical staining, Expressing, Staining, Western Blot

Journal: Cell division

Article Title: Long non-coding RNA NEAT1 induced by BHLHE40 activates Wnt/β-catenin signaling and potentiates colorectal cancer progression.

doi: 10.1186/s13008-024-00129-7

Figure Lengend Snippet: Fig. 5 BHLHE40 mediates NEAT1 transcription to activate Wnt/β-catenin signaling in CRC cells. A Western blot for the E-cadherin, N-cadherin, Vimentin, Wnt, β-catenin, c-myc, and cyclin D1 protein expression in CRC cells in response to oe-BHLHE40 (oe-NC as control) or oe-BHLHE40 + sh-NEAT1 (oe-BHLHE40 + sh-NC as control). B Effects of oe-BHLHE40 and iCRT3 combined treatment on Wnt/β-catenin pathway activity in CRC cells was measured using TOP/FOP flash assay. C MTT assay for LoVo and HCT-15 cell viability. D Transwell assay for LoVo and HCT-15 cell migration ability. E Transwell assay for LoVo and HCT-15 cell invasion ability. F Flow cytometry for LoVo and HCT-15 cell apoptosis. Results were expressed as magnitude of relative expression (means ± SD) from three independent experiments. Two-way ANOVA. *p < 0.05

Article Snippet: Afterward, the membranes were sealed with non-fat milk at room temperature for 2 h. Primary antibodies to BHLHE40 (1:1000, 17895-1-AP, ProteinTech Group, Chicago, IL, USA), Wnt (1:500, 27935-1-AP, ProteinTech Group), β-catenin (1:2000, 17565-1-AP, ProteinTech), cyclin D1 (1:5000, 26939-1-AP, ProteinTech), c-myc (1:500, GTX103436, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), E-cadherin (1:500, GTX100443, GeneTex), N-cadherin (1:500, GTX127345, GeneTex), Vimentin (1:5000, GTX100619, GeneTex), and β-actin (1:500, D110001, Sangon) were applied overnight at 4 °C.

Techniques: Western Blot, Expressing, Control, Activity Assay, MTT Assay, Transwell Assay, Migration, Flow Cytometry

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Generation of SHARP-KO cell line in TX mESCs. Top: schematic of CRISPR cut sites used to generate SHARP-KO mESCs and PCR primers used to screen for KO clones; bottom: agarose gel confirming homozygous deletion of SHARP in SHARP-KO clone H8 mESCs. (B) Schematics of constructs used to generate rescue cell lines in TX SHARP-KO or TX SHARP-HALO-AID backgrounds. Grey arrow represents dox-inducible promoter; blue box represents HALO (or eGFP) tags used; light green boxes represent RNA Recognition Motifs (RRM); wavy green line represents the Intrinsically Disordered Regions (IDRs); dark green box represents the Spen Paralog and Ortholog C-terminal (SPOC) domain. Full-length SHARP (FL-SHARP), deletion of RRM domain (ΔRRM-SHARP), deletion of IDR domain (ΔIDR-SHARP), deletion of IDR domain and insertion of alternative IDR domain from FUS protein (FUS-ΔIDR-SHARP). (C) Schematic showing experimental workflow for generating and enriching stable SHARP rescue mESCs (FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP, FUS-ΔIDR-SHARP) using constructs from . (D) Representative images of SHARP enrichment (HALO, green) over the Xi (anti-Ezh2 immunofluorescence, magenta) in female mESCs containing dox-inducible Xist, genetic deletion of SHARP, and stable integrations of HALO-tagged FL-SHARP, ΔRRM-SHARP, or ΔIDR-SHARP. Xist and SHARP rescue constructs induced with doxycycline for 72 hours; images shown as Z-sections; scale bars show 10 μm. (E) Diagram of image analysis workflow for quantifying SHARP enrichment over the Xi .

Article Snippet: Primary antibodies and the dilutions used are as follows: anti-Halo (mouse, Promega G9211, 1:200); anti-Ezh2 (mouse, Cell signaling AC22 3147S, 1:500); anti-SHARP (rabbit, Bethyl A301-119A, 1:200).

Techniques: CRISPR, Clone Assay, Agarose Gel Electrophoresis, Construct, Immunofluorescence

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Representative images of SHARP enrichment (eGFP, green) over the Xi (anti-Ezh2 immunofluorescence, magenta) in TX SHARP-KO mESCs containing dox-inducible Xist, genetic deletion of SHARP, and stable integrations of: eGFP-FL-SHARP, eGFP-ΔRRM-SHARP, eGFP-ΔIDR-SHARP, or FUS-mCherry-ΔIDR-SHARP constructs (see for cell lines details). Xist induction for 72h, images shown as z-sections; scale bars show 10 μm. (B) Quantification of images (Fig. 3A) plotting (top panel) SHARP fluorescent intensity over the Xi (denoted by Ezh2) normalized to the fluorescent intensity of a random nuclear region of the same size or (bottom panel) Ezh2 fluorescent intensity over the same area normalized to random nuclear region (see for quantification details). Values for individual nuclei (n>10) are shown; red lines represent median values; 0 represents enrichment not higher than measured over a random nuclear region. (C) SHARP enrichment across the first exon of Xist after UV-crosslinking and purification using the HALO tag in female TX SHARP-AID mESCs treated with auxin. Halo-tags were fused to FL-SHARP (top), ΔIDR-SHARP (middle), or ΔRRM-SHARP (bottom, see for cell line details). Two replicates are shown for each cell line; magenta square represents beginning of the first Xist exon, pink square demarcates A-repeat (SHARP binding site). (D) Crosslink-induced truncation sites are shown for a zoom-in on the A-repeat region from demarcated by pink square across three conditions.

Article Snippet: Primary antibodies and the dilutions used are as follows: anti-Halo (mouse, Promega G9211, 1:200); anti-Ezh2 (mouse, Cell signaling AC22 3147S, 1:500); anti-SHARP (rabbit, Bethyl A301-119A, 1:200).

Techniques: Immunofluorescence, Construct, Purification, Binding Assay

Journal: iScience

Article Title: Basic-helix-loop-helix family member e41 suppresses osteoclastogenesis and abnormal bone resorption disease via NFATc1.

doi: 10.1016/j.isci.2024.109059

Figure Lengend Snippet: Figure 3. BHLHE41 regulates osteoclast differentiation via NFATc1 (A) Heatmap of differentially expressed genes (DEGs) for BMMs in si-NC and si-BHLHE41 groups (n = 4). (B and C)Volcano plot and quantification of RNA-seq data of BMMs in si-NC and si-BHLHE41 groups. (D) GOBubble plot of KEGG enrichment analysis in DEGs. (E) GOBubble plot of GO enrichment analysis in DEGs. (F) GOCircle plot of GO enrichment analysis of the DEGs.

Article Snippet: The primary antibodies included: BHLHE41 (Affinity, Cat#AF0442, 1:1000) , NFATc1 (Affinity, Cat#DF6446, 1:1000) or GAPDH (Proteintech Group In, Cat# 60004-1-Ig, 1:4000).

Techniques: RNA Sequencing

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Schematic graph of animal sensitization and challenge. B Histological analysis of lung tissues stained with HE staining or PAS staining. C Histology score of HE staining and PAS staining. Statistical significance was determined with Mann-Whitney test. D mRNA expression of Bhlhe40 was assayed by RT-qPCR. Statistical analysis was performed by two-tailed Student’s t-test. E Western blot analysis of BHLBE40 in lung tissue. Statistical analysis was performed by two-tailed Student’s t-test. F Immunohistochemistry staining was performed to detected the level of BHLHE40 in lung tissue. Statistical analysis was performed by two-tailed Student’s t-test. G Double immunofluorescence staining for BHLHE40 (red) and F4/80 (green) in lung tissues. The number of double positive (F4/80 + BHLHE40 + ) cells was quantified. Statistical analysis was performed by two-tailed Student’s t-test. Error bars represent standard deviation. N = 8 biological replicates.

Article Snippet: The membrane was blocked in solution and incubated at 4 °C overnight with the following primary antibodies: anti- BHLHE40 (1:1000, A6534, ABclonal), anti-ARG1 (1:1000, A1847, ABclonal), anti-CD206 (1:1000, DF4149, Affinity), anti-NRTN (1:1000, DF13481, Affinity).

Techniques: Staining, MANN-WHITNEY, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Immunohistochemistry, Double Immunofluorescence Staining, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Mice in asthma group received lentiviral delivery of LV- shBhlhe40 or LV-shNC. B RT-qPCR tested the mRNA expression of Bhlhe40 in lung tissues. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. C Representative western blot and quantitative analysis of BHLHE40 expression in lung tissues. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. D Representative HE images in lung tissue sections and quantitative analysis of histologic scoring. Statistical analysis was performed by Kruskal-Wallis test. E Representative PAS images in lung tissue sections and quantitative analysis of histologic scoring. Statistical analysis was performed by Kruskal-Wallis test. F Total protein concentrations in BALF in OVA-induced asthma. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation. N = 8 biological replicates.

Article Snippet: The membrane was blocked in solution and incubated at 4 °C overnight with the following primary antibodies: anti- BHLHE40 (1:1000, A6534, ABclonal), anti-ARG1 (1:1000, A1847, ABclonal), anti-CD206 (1:1000, DF4149, Affinity), anti-NRTN (1:1000, DF13481, Affinity).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A ELISA forYM1 in BALF. B – E RT-qPCR assay showed the mRNA expression of Ym1 , Fizz1 , Cd206 , Arg1 in lung tissues. F The protein level of CD206 and ARG1 in lung tissues. G Quantitative analysis of CD206 protein level. H Quantitative analysis of ARG1 protein level. I Western blot assessed the level of BHLHE40, CD206, and ARG1 in monocyte-derived alveolar macrophages (Mo-AMs). J Quantitative analysis of BHLHE40 protein level in Mo-AMs. K Quantitative analysis of CD206 protein level in Mo-AMs. L Quantitative analysis of ARG1 protein level in Mo-AMs. N = 8 biological replicates. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation.

Article Snippet: The membrane was blocked in solution and incubated at 4 °C overnight with the following primary antibodies: anti- BHLHE40 (1:1000, A6534, ABclonal), anti-ARG1 (1:1000, A1847, ABclonal), anti-CD206 (1:1000, DF4149, Affinity), anti-NRTN (1:1000, DF13481, Affinity).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot, Derivative Assay, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Bone marrow derived macrophages (BMDMs) were isolated from BALB/c mice, and then stimulated with IL-4 to induce M1 polarization. Lentivirus infection was conducted before IL-4 treatment. B The expression level of Bhlhe40 was determined by RT-qPCR. C Western blot analysis of BHLBE40 in BMDMs. D – G The mRNA expression level of Ym1 , Fizz1 , Cd206 , and Arg1 in BMDMs. H The protein level of CD206 and ARG1 in BMDMs. I Immunofluorescence analysis of ARG1 expression in BMDMs, and the quantification of ARG1 positive cell count. N = 4 biological replicates. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation.

Article Snippet: The membrane was blocked in solution and incubated at 4 °C overnight with the following primary antibodies: anti- BHLHE40 (1:1000, A6534, ABclonal), anti-ARG1 (1:1000, A1847, ABclonal), anti-CD206 (1:1000, DF4149, Affinity), anti-NRTN (1:1000, DF13481, Affinity).

Techniques: Derivative Assay, Isolation, Infection, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Cell Counting, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A , B Nrtn expression level was determined by RT-qPCR and western blot. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. C Dual-luciferase reporter assay. N = 3 biological replicates. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. D Validation by ChIP-qPCR analysis of BHLHE40 binding to Nrtn in BMDMs. Statistical analysis was performed by two-tailed Student’s t-test. E RT-qPCR showed the mRNA expression of Fizz1 , Ym1 , Arg1 , and Cd206 . Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. F The protein level of CD206 and ARG1 was assayed by western blot. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. G Immunofluorescence analysis of ARG1 expression in BMDMs, and the quantification of ARG1 positive cell count. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation. N = 3-4 biological replicates.

Article Snippet: The membrane was blocked in solution and incubated at 4 °C overnight with the following primary antibodies: anti- BHLHE40 (1:1000, A6534, ABclonal), anti-ARG1 (1:1000, A1847, ABclonal), anti-CD206 (1:1000, DF4149, Affinity), anti-NRTN (1:1000, DF13481, Affinity).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay, Biomarker Discovery, ChIP-qPCR, Binding Assay, Two Tailed Test, Immunofluorescence, Cell Counting, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Mice were stimulated with thioglycolate and IL-4 by intraperitoneal injection, and then physiological peritoneal macrophages were obtained. B Validation by ChIP-qPCR analysis of BHLHE40 binding to Nrtn in peritoneal macrophages. Statistical analysis was performed by two-tailed Student’s t-test. C RT-qPCR examined the mRNA level of Bhlhe40 in peritoneal macrophages. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. D Representative immunoblot and quantitative analysis of BHLHE40 expression. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. E Immunofluorescence analysis of ARG1 expression in peritoneal macrophages, and the quantification of ARG1 positive cell count. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. F Peritoneal macrophages were infected with LV- shBhlhe40 and LV- shNrtn , and then were subjected to immunofluorescence staining for ARG1. The number of ARG1 positive cells was quantified. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation. N = 4 biological replicates.

Article Snippet: The membrane was blocked in solution and incubated at 4 °C overnight with the following primary antibodies: anti- BHLHE40 (1:1000, A6534, ABclonal), anti-ARG1 (1:1000, A1847, ABclonal), anti-CD206 (1:1000, DF4149, Affinity), anti-NRTN (1:1000, DF13481, Affinity).

Techniques: Injection, Biomarker Discovery, ChIP-qPCR, Binding Assay, Two Tailed Test, Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Cell Counting, Infection, Staining, Standard Deviation

Journal: Cell reports

Article Title: TFEB Transcriptional Responses Reveal Negative Feedback by BHLHE40 and BHLHE41

doi: 10.1016/j.celrep.2020.108371

Figure Lengend Snippet: (A) Volcano plots depicting differential gene expression in BHLHE40/41-WT versus BHLHE40/41-KO cells reconstituted cells at steady state. Highlighted in red (left panel) are genes significantly upregulated in TFEB-WT versus TFEB-KO cells following Torin treatment (logFC > ln4 and q < 0.01). Highlighted in cyan (right panel) are select TFEB target genes.

Article Snippet: BHLHE40 Human Tagged ORF clone , OriGene , RC210294L1.

Techniques: Gene Expression

Journal: Cell reports

Article Title: TFEB Transcriptional Responses Reveal Negative Feedback by BHLHE40 and BHLHE41

doi: 10.1016/j.celrep.2020.108371

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: BHLHE40 Human Tagged ORF clone , OriGene , RC210294L1.

Techniques: Binding Assay, Virus, Recombinant, Electron Microscopy, Staining, cDNA Synthesis, SYBR Green Assay, Software, Imaging

Journal: eLife

Article Title: BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

doi: 10.7554/elife.36826

Figure Lengend Snippet: Figure 1. Co-immunoprecipitation of BHLHE40 phosphorylated at serine-383 (S383). (A) Protocol for identifying the phosphoproteins that are induced by feeding and interact with both LXRa and C/EBPb. Rats were either fasted for 48 hr, or fasted for 48 hr and then refed with a high-carbohydrate diet for 6 hr. Nuclear extracts were prepared from livers and subjected to immunoprecipitation with anti-LXRa or with anti-C/EBPb, after which the immunoprecipitates were subjected to 10% SDS-PAGE and analyzed by mass spectrometry. Phosphoproteins that were enriched (threefold or greater) in refed samples and co-immunoprecipitated with both anti-LXRa and anti-C/EBPb were identified. (B) LC-MS/MS analysis of immunoprecipitated proteins. (C) Identification of BHLHE40 peptides. The intensity ratio (refed/fasted) was calculated using the summed intensity of all peptides in the protein. (D) MS/MS spectra data of BHLHE40 phosphorylated at Ser-383. LC-MS/MS sample preparation and analysis are described in Materials and methods. DOI: https://doi.org/10.7554/eLife.36826.002

Article Snippet: Description Source or reference Identifier 3,30,5-Triiodo-L-thyronine Sigma-Aldrich, St. Louis, MO T2877 Bovine insulin Sigma-Aldrich I6634 Bio-Gen PRO 200 Homogenizer PRO Scientific, Oxford, CT 01–01200 Calpain inhibitor I (ALLN) AG Scientific, San Diego, CA CAS110044-82-1 Chromatin Immunoprecipitation (ChIP) Assay Kit EMD Millipore Corp, Billerica, MA 17–295 Collagen I-coated dishes BD Sciences, Franklin Lakes, NJ 356400 Dexamethasone Sigma-Aldrich D4902 DMEM Sigma-Aldrich D6046 Freund’s Adjuvant, Incomplete Sigma-Aldrich F5506 Halt Phosphatase Inhibitor Cocktail Thermo Fisher Scientific, Waltham, MA 78426 High-Carbohydrate/Fat-free Diet MP Biomedicals, Santa Ana, CA 960238 Lentiviurs Encoding Rat BHLHE40 Origene, Rockville, MD BR206868LV Lipofectamine 2000 Invitrogen, Carlsbad, CA 11668–027 Medium 199 Invitrogen 11150–059 nProtein A Sepharose 4 Fast Flow Affinity Media GE Healthcare, Chicago, IL 17-5280-04 PBS Sigma-Aldrich D8537 Phenylmethysulfonyl fluoride (PMSF) Sigma-Aldrich P7626 Pierce Co-Immuno- precipitation Kit Thermo Fisher Scientific 26149 Protease Inhibitor cOmplete Tablets Roche Holding AG, Basel, CH 5892791001 Proteinase K Thermo Fisher Scientific AM2546 ProteoSilver Stain Kit Sigma-Aldrich PROTSIL1 Rapamycin Sigma-Aldrich R0395 Teklad Global 18% Protein Rodent Diet (chow diet for mice) Harlan Laboratories, Indianapolis, IN 2018 Teklad Global 16% Protein Rodent Diet (chow diet for rats) Harlan Laboratories 2016 Tian et al. eLife 2018;7:e36826.

Techniques: Immunoprecipitation, SDS Page, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Sample Prep

Journal: eLife

Article Title: BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

doi: 10.7554/elife.36826

Figure Lengend Snippet: Figure 2. Relative amounts of mRNA for BHLHE40 (A), SREBP-1c (B), LXRa (C), and C/EBPb (D), in livers of rats subjected to fasting and refeeding. Male rats (age 2–3 months) were either fasted for 48 hr, or fasted for 48 hr and then refed with a high-carbohydrate diet for 6 hr. Total liver RNA from eight rats in each group was subjected to quantitative RT-PCR. Each black circle (fasted rats) or red triangle (refed rats) represents an individual animal. Each value in the refed group represents the amount of mRNA relative to the mean amount in the fasted group, which is arbitrarily defined as 1.0. Mean Ct values for BHLHE40, SREBP-1c, LXRa, and C/EBPb in the fasted groups were 24.9, 23.9, 24.9, and 24.4, respectively. NS, not significant. p-Values calculated using Student’s t-test. DOI: https://doi.org/10.7554/eLife.36826.003

Article Snippet: Description Source or reference Identifier 3,30,5-Triiodo-L-thyronine Sigma-Aldrich, St. Louis, MO T2877 Bovine insulin Sigma-Aldrich I6634 Bio-Gen PRO 200 Homogenizer PRO Scientific, Oxford, CT 01–01200 Calpain inhibitor I (ALLN) AG Scientific, San Diego, CA CAS110044-82-1 Chromatin Immunoprecipitation (ChIP) Assay Kit EMD Millipore Corp, Billerica, MA 17–295 Collagen I-coated dishes BD Sciences, Franklin Lakes, NJ 356400 Dexamethasone Sigma-Aldrich D4902 DMEM Sigma-Aldrich D6046 Freund’s Adjuvant, Incomplete Sigma-Aldrich F5506 Halt Phosphatase Inhibitor Cocktail Thermo Fisher Scientific, Waltham, MA 78426 High-Carbohydrate/Fat-free Diet MP Biomedicals, Santa Ana, CA 960238 Lentiviurs Encoding Rat BHLHE40 Origene, Rockville, MD BR206868LV Lipofectamine 2000 Invitrogen, Carlsbad, CA 11668–027 Medium 199 Invitrogen 11150–059 nProtein A Sepharose 4 Fast Flow Affinity Media GE Healthcare, Chicago, IL 17-5280-04 PBS Sigma-Aldrich D8537 Phenylmethysulfonyl fluoride (PMSF) Sigma-Aldrich P7626 Pierce Co-Immuno- precipitation Kit Thermo Fisher Scientific 26149 Protease Inhibitor cOmplete Tablets Roche Holding AG, Basel, CH 5892791001 Proteinase K Thermo Fisher Scientific AM2546 ProteoSilver Stain Kit Sigma-Aldrich PROTSIL1 Rapamycin Sigma-Aldrich R0395 Teklad Global 18% Protein Rodent Diet (chow diet for mice) Harlan Laboratories, Indianapolis, IN 2018 Teklad Global 16% Protein Rodent Diet (chow diet for rats) Harlan Laboratories 2016 Tian et al. eLife 2018;7:e36826.

Techniques: Quantitative RT-PCR

Journal: eLife

Article Title: BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

doi: 10.7554/elife.36826

Figure Lengend Snippet: Figure 3. Co-immunoprecipitation of LXRa, C/EBPb, and BHLHE40 in livers from fasted and refed rats. Nuclear extracts were prepared from livers of rats subjected to 48 hr fasting (F) or 48 hr fasting followed by 6 hr refeeding (R). Immunoprecipitations were carried out with agarose beads conjugated with the indicated antibody as described in Materials and methods. After centrifugation, the beads were washed and boiled in SDS loading buffer to elute the proteins. Aliquots of the input (30% of total) and eluates from the immunoprecipitations (100% of eluated fraction) were subjected to SDS-PAGE and immunoblotted with the indicated antibodies as follows: 2 mg/ml of polyclonal anti-LXRa, a 1:1000 dilution of polyclonal anti-C/EBPb, 1 mg/ml of polyclonal anti-BHLHE40, 1 mg/ml of monoclonal anti-SREBP-1, or a 1:1000 dilution of monoclonal anti-CREB. Proteins were detected with the LI-COR Odessy Infrared Imaging System using a 1:5000 dilution of anti-rabbit IgG conjugated to horseradish peroxidase. DOI: https://doi.org/10.7554/eLife.36826.004

Article Snippet: Description Source or reference Identifier 3,30,5-Triiodo-L-thyronine Sigma-Aldrich, St. Louis, MO T2877 Bovine insulin Sigma-Aldrich I6634 Bio-Gen PRO 200 Homogenizer PRO Scientific, Oxford, CT 01–01200 Calpain inhibitor I (ALLN) AG Scientific, San Diego, CA CAS110044-82-1 Chromatin Immunoprecipitation (ChIP) Assay Kit EMD Millipore Corp, Billerica, MA 17–295 Collagen I-coated dishes BD Sciences, Franklin Lakes, NJ 356400 Dexamethasone Sigma-Aldrich D4902 DMEM Sigma-Aldrich D6046 Freund’s Adjuvant, Incomplete Sigma-Aldrich F5506 Halt Phosphatase Inhibitor Cocktail Thermo Fisher Scientific, Waltham, MA 78426 High-Carbohydrate/Fat-free Diet MP Biomedicals, Santa Ana, CA 960238 Lentiviurs Encoding Rat BHLHE40 Origene, Rockville, MD BR206868LV Lipofectamine 2000 Invitrogen, Carlsbad, CA 11668–027 Medium 199 Invitrogen 11150–059 nProtein A Sepharose 4 Fast Flow Affinity Media GE Healthcare, Chicago, IL 17-5280-04 PBS Sigma-Aldrich D8537 Phenylmethysulfonyl fluoride (PMSF) Sigma-Aldrich P7626 Pierce Co-Immuno- precipitation Kit Thermo Fisher Scientific 26149 Protease Inhibitor cOmplete Tablets Roche Holding AG, Basel, CH 5892791001 Proteinase K Thermo Fisher Scientific AM2546 ProteoSilver Stain Kit Sigma-Aldrich PROTSIL1 Rapamycin Sigma-Aldrich R0395 Teklad Global 18% Protein Rodent Diet (chow diet for mice) Harlan Laboratories, Indianapolis, IN 2018 Teklad Global 16% Protein Rodent Diet (chow diet for rats) Harlan Laboratories 2016 Tian et al. eLife 2018;7:e36826.

Techniques: Immunoprecipitation, Centrifugation, SDS Page, Imaging

Journal: eLife

Article Title: BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

doi: 10.7554/elife.36826

Figure Lengend Snippet: Figure 4. ChIP assay to demonstrate BHLHE40 binding to the enhancer/promoter region of rat SREBP-1c gene. (A) Schematic diagram of rat SREBP-1c enhancer/promoter region, showing the location of the two LXREs. Arrows denote the location of the primers used in the ChIP assays. (B) ChIP assay was performed with livers from rats that had been subjected to either 48 hr fasting (F) or 48 hr fasting followed by 6 hr refeeding (R), as described in Materials and methods. After crosslinking, the sheared chromatin was incubated with 5 mg/mL of one of the following antibodies: anti-IgG (control), anti-BHLHE40, anti-LXRa, or anti-C/EBPb, after which the DNA was purified from each immunoprecipitate and subjected to PCR with primer pairs 1 or 2, and the products visualized on an agarose gel. The DNA sequences for primers 1 and 2 are described in Table 2 in the paper by Tian et al. (2016). DOI: https://doi.org/10.7554/eLife.36826.005

Article Snippet: Description Source or reference Identifier 3,30,5-Triiodo-L-thyronine Sigma-Aldrich, St. Louis, MO T2877 Bovine insulin Sigma-Aldrich I6634 Bio-Gen PRO 200 Homogenizer PRO Scientific, Oxford, CT 01–01200 Calpain inhibitor I (ALLN) AG Scientific, San Diego, CA CAS110044-82-1 Chromatin Immunoprecipitation (ChIP) Assay Kit EMD Millipore Corp, Billerica, MA 17–295 Collagen I-coated dishes BD Sciences, Franklin Lakes, NJ 356400 Dexamethasone Sigma-Aldrich D4902 DMEM Sigma-Aldrich D6046 Freund’s Adjuvant, Incomplete Sigma-Aldrich F5506 Halt Phosphatase Inhibitor Cocktail Thermo Fisher Scientific, Waltham, MA 78426 High-Carbohydrate/Fat-free Diet MP Biomedicals, Santa Ana, CA 960238 Lentiviurs Encoding Rat BHLHE40 Origene, Rockville, MD BR206868LV Lipofectamine 2000 Invitrogen, Carlsbad, CA 11668–027 Medium 199 Invitrogen 11150–059 nProtein A Sepharose 4 Fast Flow Affinity Media GE Healthcare, Chicago, IL 17-5280-04 PBS Sigma-Aldrich D8537 Phenylmethysulfonyl fluoride (PMSF) Sigma-Aldrich P7626 Pierce Co-Immuno- precipitation Kit Thermo Fisher Scientific 26149 Protease Inhibitor cOmplete Tablets Roche Holding AG, Basel, CH 5892791001 Proteinase K Thermo Fisher Scientific AM2546 ProteoSilver Stain Kit Sigma-Aldrich PROTSIL1 Rapamycin Sigma-Aldrich R0395 Teklad Global 18% Protein Rodent Diet (chow diet for mice) Harlan Laboratories, Indianapolis, IN 2018 Teklad Global 16% Protein Rodent Diet (chow diet for rats) Harlan Laboratories 2016 Tian et al. eLife 2018;7:e36826.

Techniques: Binding Assay, Incubation, Control, Purification, Agarose Gel Electrophoresis

Journal: eLife

Article Title: BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

doi: 10.7554/elife.36826

Figure Lengend Snippet: Figure 6. Overexpression of BHLHE40 fails to overcome rapamycin-mediated inhibition of SREBP-1c induction by insulin in primary rat hepatocytes. Rat hepatocytes were prepared and plated on day 0 as described in Materials and methods. At 2 hr after plating, cells were infected with or without lentivirus expressing rat BHLHE40 (Lenti- B40; 5 105 TU/well) as indicated. On day 1, cells were pretreated with or without 100 nM rapamycin for 30 min, after which the cells received either no insulin or 100 nM insulin for 6 hr as indicated. The cells were then harvested, and the levels of mRNA encoding BHLHE40 (A) and SREBP-1c (B) were determined by RT-PCR. Each dot represents a value from an individual dish. Mean Ct values for BHLHE40 and SREBP-1c in the untreated control groups were 22.8 and 25.6, respectively. DOI: https://doi.org/10.7554/eLife.36826.007

Article Snippet: Description Source or reference Identifier 3,30,5-Triiodo-L-thyronine Sigma-Aldrich, St. Louis, MO T2877 Bovine insulin Sigma-Aldrich I6634 Bio-Gen PRO 200 Homogenizer PRO Scientific, Oxford, CT 01–01200 Calpain inhibitor I (ALLN) AG Scientific, San Diego, CA CAS110044-82-1 Chromatin Immunoprecipitation (ChIP) Assay Kit EMD Millipore Corp, Billerica, MA 17–295 Collagen I-coated dishes BD Sciences, Franklin Lakes, NJ 356400 Dexamethasone Sigma-Aldrich D4902 DMEM Sigma-Aldrich D6046 Freund’s Adjuvant, Incomplete Sigma-Aldrich F5506 Halt Phosphatase Inhibitor Cocktail Thermo Fisher Scientific, Waltham, MA 78426 High-Carbohydrate/Fat-free Diet MP Biomedicals, Santa Ana, CA 960238 Lentiviurs Encoding Rat BHLHE40 Origene, Rockville, MD BR206868LV Lipofectamine 2000 Invitrogen, Carlsbad, CA 11668–027 Medium 199 Invitrogen 11150–059 nProtein A Sepharose 4 Fast Flow Affinity Media GE Healthcare, Chicago, IL 17-5280-04 PBS Sigma-Aldrich D8537 Phenylmethysulfonyl fluoride (PMSF) Sigma-Aldrich P7626 Pierce Co-Immuno- precipitation Kit Thermo Fisher Scientific 26149 Protease Inhibitor cOmplete Tablets Roche Holding AG, Basel, CH 5892791001 Proteinase K Thermo Fisher Scientific AM2546 ProteoSilver Stain Kit Sigma-Aldrich PROTSIL1 Rapamycin Sigma-Aldrich R0395 Teklad Global 18% Protein Rodent Diet (chow diet for mice) Harlan Laboratories, Indianapolis, IN 2018 Teklad Global 16% Protein Rodent Diet (chow diet for rats) Harlan Laboratories 2016 Tian et al. eLife 2018;7:e36826.

Techniques: Over Expression, Inhibition, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: eLife

Article Title: BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

doi: 10.7554/elife.36826

Figure Lengend Snippet: Figure 5. Requirement for mTOR in induction in mRNAs encoding SREBP-1c (A) and BHLHE40 (B) in livers of rats subjected to fasting and refeeding. Sixteen male rats (age 2–3 months) were fasted for 48 hr. Six hours prior to sacrifice, eight rats received an intraperitoneal injection of 20 mg/kg rapamycin, and the other eight received vehicle. Four of the animals in the treated and untreated groups continued to fast, and the other four rats were refed a high-carbohydrate diet as described in Materials and methods. After 6 hr, the rats were sacrificed, and livers were homogenized. Equal amounts of total RNA from the livers of the four rats in each group were pooled and subjected to quantitative RT-PCR. Each bar represents the amount of mRNA relative to that of the vehicle- treated fasted group, which was defined as 1.0. Ct values for BHLHE40 and SREBP-1c in the fasted and vehicle treated group were 24.0 and 23.7, respectively. DOI: https://doi.org/10.7554/eLife.36826.006

Article Snippet: Description Source or reference Identifier 3,30,5-Triiodo-L-thyronine Sigma-Aldrich, St. Louis, MO T2877 Bovine insulin Sigma-Aldrich I6634 Bio-Gen PRO 200 Homogenizer PRO Scientific, Oxford, CT 01–01200 Calpain inhibitor I (ALLN) AG Scientific, San Diego, CA CAS110044-82-1 Chromatin Immunoprecipitation (ChIP) Assay Kit EMD Millipore Corp, Billerica, MA 17–295 Collagen I-coated dishes BD Sciences, Franklin Lakes, NJ 356400 Dexamethasone Sigma-Aldrich D4902 DMEM Sigma-Aldrich D6046 Freund’s Adjuvant, Incomplete Sigma-Aldrich F5506 Halt Phosphatase Inhibitor Cocktail Thermo Fisher Scientific, Waltham, MA 78426 High-Carbohydrate/Fat-free Diet MP Biomedicals, Santa Ana, CA 960238 Lentiviurs Encoding Rat BHLHE40 Origene, Rockville, MD BR206868LV Lipofectamine 2000 Invitrogen, Carlsbad, CA 11668–027 Medium 199 Invitrogen 11150–059 nProtein A Sepharose 4 Fast Flow Affinity Media GE Healthcare, Chicago, IL 17-5280-04 PBS Sigma-Aldrich D8537 Phenylmethysulfonyl fluoride (PMSF) Sigma-Aldrich P7626 Pierce Co-Immuno- precipitation Kit Thermo Fisher Scientific 26149 Protease Inhibitor cOmplete Tablets Roche Holding AG, Basel, CH 5892791001 Proteinase K Thermo Fisher Scientific AM2546 ProteoSilver Stain Kit Sigma-Aldrich PROTSIL1 Rapamycin Sigma-Aldrich R0395 Teklad Global 18% Protein Rodent Diet (chow diet for mice) Harlan Laboratories, Indianapolis, IN 2018 Teklad Global 16% Protein Rodent Diet (chow diet for rats) Harlan Laboratories 2016 Tian et al. eLife 2018;7:e36826.

Techniques: Injection, Quantitative RT-PCR

Journal: eLife

Article Title: BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

doi: 10.7554/elife.36826

Figure Lengend Snippet: Figure 7. Time course of induction of BHLHE40 and SREBP-1c mRNA in livers of refed rats (A–C) and in primary rat hepatocytes treated with insulin (D–F). (A–C) Male rats (age, 2–3 months) were fasted for 48 hr and then refed with a high-carbohydrate diet for the indicated time, after which total RNA from the liver was subjected to quantitative RT-PCR. Each point in (A) and (B) represents the mRNA level from a single animal relative to the mean value from three fasted rats (i.e. zero-time values). Zero-time Ct values for BHLHE40 and SREBP-1c were 23.5 and 25.7, respectively. Values in (C) represent the mean ± SEM of the values from (A) and (B) plotted as the percentages of the 6 hr value, which is defined as 100%. (D–F) Hepatocytes from nonfasted male rats (age, 2–3 months) on a chow diet were prepared and plated on day 0. On day 1, the cells received either no insulin or 100 nM insulin for the indicated time, after which the cells were harvested for measurement of total RNAs by quantitative RT-PCR. Each value in (D) and (E) (6 hr time course) represents the amount of mRNA from a single dish relative to that of the mean value from the three dishes at zero-time, which is defined as 1.0. Mean Ct values (zero-time) for BHLHE40 and SREBP-1c in the absence of insulin were 23.6 and 26.4, respectively. The values in (F) (1 hr time course) represent the mean ±SEM of the values from three dishes. Mean Ct values (zero-time) for BHLHE40, SREBP-1c, and PEPCK in the absence of insulin were 23.6, 26.3, and 20.0, respectively. DOI: https://doi.org/10.7554/eLife.36826.008

Article Snippet: Description Source or reference Identifier 3,30,5-Triiodo-L-thyronine Sigma-Aldrich, St. Louis, MO T2877 Bovine insulin Sigma-Aldrich I6634 Bio-Gen PRO 200 Homogenizer PRO Scientific, Oxford, CT 01–01200 Calpain inhibitor I (ALLN) AG Scientific, San Diego, CA CAS110044-82-1 Chromatin Immunoprecipitation (ChIP) Assay Kit EMD Millipore Corp, Billerica, MA 17–295 Collagen I-coated dishes BD Sciences, Franklin Lakes, NJ 356400 Dexamethasone Sigma-Aldrich D4902 DMEM Sigma-Aldrich D6046 Freund’s Adjuvant, Incomplete Sigma-Aldrich F5506 Halt Phosphatase Inhibitor Cocktail Thermo Fisher Scientific, Waltham, MA 78426 High-Carbohydrate/Fat-free Diet MP Biomedicals, Santa Ana, CA 960238 Lentiviurs Encoding Rat BHLHE40 Origene, Rockville, MD BR206868LV Lipofectamine 2000 Invitrogen, Carlsbad, CA 11668–027 Medium 199 Invitrogen 11150–059 nProtein A Sepharose 4 Fast Flow Affinity Media GE Healthcare, Chicago, IL 17-5280-04 PBS Sigma-Aldrich D8537 Phenylmethysulfonyl fluoride (PMSF) Sigma-Aldrich P7626 Pierce Co-Immuno- precipitation Kit Thermo Fisher Scientific 26149 Protease Inhibitor cOmplete Tablets Roche Holding AG, Basel, CH 5892791001 Proteinase K Thermo Fisher Scientific AM2546 ProteoSilver Stain Kit Sigma-Aldrich PROTSIL1 Rapamycin Sigma-Aldrich R0395 Teklad Global 18% Protein Rodent Diet (chow diet for mice) Harlan Laboratories, Indianapolis, IN 2018 Teklad Global 16% Protein Rodent Diet (chow diet for rats) Harlan Laboratories 2016 Tian et al. eLife 2018;7:e36826.

Techniques: Quantitative RT-PCR

Journal: eLife

Article Title: BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

doi: 10.7554/elife.36826

Figure Lengend Snippet: Figure 8. Knockdown of BHLHE40 mRNA in rat hepatocytes reduces insulin-induced mRNA for SREBP-1c, but not mRNA for LXRa or C/EBPb. Hepatocytes from non-fasted male rats (age, 2–3 months) on a chow diet were prepared and plated on day 0. After attachment for 2 hr, cells were transfected with either 50 nM control siRNA (siControl) or one of two siRNAs targeting BHLHE40 (siB40-A or siB40-B). On day 1, cells were treated with or without 100 nM insulin for 6 hr and then harvested for measurement of the indicated mRNA by quantitative RT- PCR. Each value represents an individual dish. Mean Ct values in the absence of insulin for BHLHE40, SREBP-1c, LXRa, and C/EBPb were 21.8, 24.7, 25.5, and 22.2, respectively. DOI: https://doi.org/10.7554/eLife.36826.009

Article Snippet: Description Source or reference Identifier 3,30,5-Triiodo-L-thyronine Sigma-Aldrich, St. Louis, MO T2877 Bovine insulin Sigma-Aldrich I6634 Bio-Gen PRO 200 Homogenizer PRO Scientific, Oxford, CT 01–01200 Calpain inhibitor I (ALLN) AG Scientific, San Diego, CA CAS110044-82-1 Chromatin Immunoprecipitation (ChIP) Assay Kit EMD Millipore Corp, Billerica, MA 17–295 Collagen I-coated dishes BD Sciences, Franklin Lakes, NJ 356400 Dexamethasone Sigma-Aldrich D4902 DMEM Sigma-Aldrich D6046 Freund’s Adjuvant, Incomplete Sigma-Aldrich F5506 Halt Phosphatase Inhibitor Cocktail Thermo Fisher Scientific, Waltham, MA 78426 High-Carbohydrate/Fat-free Diet MP Biomedicals, Santa Ana, CA 960238 Lentiviurs Encoding Rat BHLHE40 Origene, Rockville, MD BR206868LV Lipofectamine 2000 Invitrogen, Carlsbad, CA 11668–027 Medium 199 Invitrogen 11150–059 nProtein A Sepharose 4 Fast Flow Affinity Media GE Healthcare, Chicago, IL 17-5280-04 PBS Sigma-Aldrich D8537 Phenylmethysulfonyl fluoride (PMSF) Sigma-Aldrich P7626 Pierce Co-Immuno- precipitation Kit Thermo Fisher Scientific 26149 Protease Inhibitor cOmplete Tablets Roche Holding AG, Basel, CH 5892791001 Proteinase K Thermo Fisher Scientific AM2546 ProteoSilver Stain Kit Sigma-Aldrich PROTSIL1 Rapamycin Sigma-Aldrich R0395 Teklad Global 18% Protein Rodent Diet (chow diet for mice) Harlan Laboratories, Indianapolis, IN 2018 Teklad Global 16% Protein Rodent Diet (chow diet for rats) Harlan Laboratories 2016 Tian et al. eLife 2018;7:e36826.

Techniques: Knockdown, Transfection, Control, Quantitative RT-PCR

Journal: eLife

Article Title: BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

doi: 10.7554/elife.36826

Figure Lengend Snippet: Figure 9. Knockout of Bhlhe40 gene in mice decreases SREBP-1c mRNA in livers of refed animals. (A) Immunoblot analysis of liver nuclear extracts from wild type (WT) and Bhlhe40/ (KO) mice. Nuclear extracts (20 mg protein) were subjected to 4–12% SDS-PAGE, followed by immunoblot analysis with 1.5 mg/ml of rabbit polyclonal anti-BHLHE40 (directed against amino acids 1–60 of rat BHLHE40 protein). LSD1 (lysine-specific demethylase 1) served as a loading control and was immunoblotted with a 1:1000 dilution of rabbit monoclonal anti-LSD1. Proteins were detected with the LI-COR Odyssey Infrared Imaging System using a 1:5000 dilution of anti-rabbit IgG conjugated to horseradish peroxidase. (B and C) WT and Bhlhe40 KO mice were fasted overnight and then refed a high-carbohydrate diet for 4 hr, after which total liver RNA was prepared and subjected to quantitative RT-PCR. Each circle (WT) or triangle (KO) represents an individual mouse. The mean value for WT mice is defined as 1.0. (B) mRNAs in livers of male WT and KO mice (age 6 wk; 5 mice/group). Mean Ct values for BHLHE40, SREBP-1c, LXRa and C/EBPb in WT mice were 21.2, 22.2, 21.7, and 22.1, respectively. (C) mRNAs in livers of female WT and KO mice (age 7 wk; 7 mice/group). Mean Ct values for BHLHE40, SREBP-1c, LXRa and C/EBPb in WT mice were 21.5, 21.0, 22.3, and 22.5, respectively. p-Values calculated using Student t-test. DOI: https://doi.org/10.7554/eLife.36826.010

Article Snippet: Description Source or reference Identifier 3,30,5-Triiodo-L-thyronine Sigma-Aldrich, St. Louis, MO T2877 Bovine insulin Sigma-Aldrich I6634 Bio-Gen PRO 200 Homogenizer PRO Scientific, Oxford, CT 01–01200 Calpain inhibitor I (ALLN) AG Scientific, San Diego, CA CAS110044-82-1 Chromatin Immunoprecipitation (ChIP) Assay Kit EMD Millipore Corp, Billerica, MA 17–295 Collagen I-coated dishes BD Sciences, Franklin Lakes, NJ 356400 Dexamethasone Sigma-Aldrich D4902 DMEM Sigma-Aldrich D6046 Freund’s Adjuvant, Incomplete Sigma-Aldrich F5506 Halt Phosphatase Inhibitor Cocktail Thermo Fisher Scientific, Waltham, MA 78426 High-Carbohydrate/Fat-free Diet MP Biomedicals, Santa Ana, CA 960238 Lentiviurs Encoding Rat BHLHE40 Origene, Rockville, MD BR206868LV Lipofectamine 2000 Invitrogen, Carlsbad, CA 11668–027 Medium 199 Invitrogen 11150–059 nProtein A Sepharose 4 Fast Flow Affinity Media GE Healthcare, Chicago, IL 17-5280-04 PBS Sigma-Aldrich D8537 Phenylmethysulfonyl fluoride (PMSF) Sigma-Aldrich P7626 Pierce Co-Immuno- precipitation Kit Thermo Fisher Scientific 26149 Protease Inhibitor cOmplete Tablets Roche Holding AG, Basel, CH 5892791001 Proteinase K Thermo Fisher Scientific AM2546 ProteoSilver Stain Kit Sigma-Aldrich PROTSIL1 Rapamycin Sigma-Aldrich R0395 Teklad Global 18% Protein Rodent Diet (chow diet for mice) Harlan Laboratories, Indianapolis, IN 2018 Teklad Global 16% Protein Rodent Diet (chow diet for rats) Harlan Laboratories 2016 Tian et al. eLife 2018;7:e36826.

Techniques: Knock-Out, Western Blot, SDS Page, Control, Imaging, Quantitative RT-PCR

Journal: eLife

Article Title: BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

doi: 10.7554/elife.36826

Figure Lengend Snippet: Figure 10. AAV-mediated injection of rat BHLHE40 restores the decrease of SREBP-1c mRNA in livers of refed Bhlhe40 knockout mice. WT and KO mice (age 6 wk; 4 mice/group) received tail vein injections of AAV encoding a control mRNA (AAV-EGFP), wild-type BHLHE40, or mutant BHLHE40(S383A). Three weeks after injection, mice were fasted overnight and then refed with a high-carbohydrate diet for 4 hr, after which total liver RNA was prepared and subjected to quantitative RT-PCR. Each circle, triangle, or square represents an individual mouse. The mRNA expression is plotted as the amount relative to livers of WT mice injected with the control AAV-EGFP, which is assigned a value of 1.0. Mean Ct values for BHLHE40, SREBP-1c, LXRa and C/EBPb in the livers of WT mice receiving AAV-EGFP were 22.3, 25.4, 22.4, and 22.7, respectively. *p<0.01; **p<0.001; NS, not significant. p-Values calculated using Student t-test. DOI: https://doi.org/10.7554/eLife.36826.011

Article Snippet: Description Source or reference Identifier 3,30,5-Triiodo-L-thyronine Sigma-Aldrich, St. Louis, MO T2877 Bovine insulin Sigma-Aldrich I6634 Bio-Gen PRO 200 Homogenizer PRO Scientific, Oxford, CT 01–01200 Calpain inhibitor I (ALLN) AG Scientific, San Diego, CA CAS110044-82-1 Chromatin Immunoprecipitation (ChIP) Assay Kit EMD Millipore Corp, Billerica, MA 17–295 Collagen I-coated dishes BD Sciences, Franklin Lakes, NJ 356400 Dexamethasone Sigma-Aldrich D4902 DMEM Sigma-Aldrich D6046 Freund’s Adjuvant, Incomplete Sigma-Aldrich F5506 Halt Phosphatase Inhibitor Cocktail Thermo Fisher Scientific, Waltham, MA 78426 High-Carbohydrate/Fat-free Diet MP Biomedicals, Santa Ana, CA 960238 Lentiviurs Encoding Rat BHLHE40 Origene, Rockville, MD BR206868LV Lipofectamine 2000 Invitrogen, Carlsbad, CA 11668–027 Medium 199 Invitrogen 11150–059 nProtein A Sepharose 4 Fast Flow Affinity Media GE Healthcare, Chicago, IL 17-5280-04 PBS Sigma-Aldrich D8537 Phenylmethysulfonyl fluoride (PMSF) Sigma-Aldrich P7626 Pierce Co-Immuno- precipitation Kit Thermo Fisher Scientific 26149 Protease Inhibitor cOmplete Tablets Roche Holding AG, Basel, CH 5892791001 Proteinase K Thermo Fisher Scientific AM2546 ProteoSilver Stain Kit Sigma-Aldrich PROTSIL1 Rapamycin Sigma-Aldrich R0395 Teklad Global 18% Protein Rodent Diet (chow diet for mice) Harlan Laboratories, Indianapolis, IN 2018 Teklad Global 16% Protein Rodent Diet (chow diet for rats) Harlan Laboratories 2016 Tian et al. eLife 2018;7:e36826.

Techniques: Injection, Knock-Out, Control, Mutagenesis, Quantitative RT-PCR, Expressing