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: Molecular Cancer Research

Article Title: Diurnal Expression of PD-1 on Tumor-Associated Macrophages Underlies the Dosing Time-Dependent Antitumor Effects of the PD-1/PD-L1 Inhibitor BMS-1 in B16/BL6 Melanoma-Bearing Mice

doi: 10.1158/1541-7786.mcr-21-0786

Figure Lengend Snippet: Figure 1. Diurnal variations in the population rate of PD-1–expressing TAMs in mouse B16/BL6 melanoma-forming tumor masses. A, Schematic depicting the isolation method of TAMs from mouse B16/BL6 melanoma-forming tumor masses. B, Temporal mRNA expression profiles of Bmal1, Clock, Per1, Per2, Cry1, Dec1, Dec2, Dbp, Nfil3, Rora, and Rev-erba in TAMs and circulating monocytes. Data were normalized by 18S rRNA levels. Values are the mean with SD (n ¼ 3). There were significant time-dependent variations in the mRNA levels of all circadian clock gene in both TAMs and circulating monocytes (P < 0.01, respectively; one-way ANOVA). C, The left diagrams show the representative proportion of PD-1þ TAMs collected at ZT6 and ZT18. Right panel shows temporal profiles of the population of PD-1–expressing F4/80þ CD11bþ CD206þ TAMs. Values are the mean with SD (n ¼ 3). There was a significant time-dependent variation in the population of PD-1–expressing TAMs. (F5,12 ¼ 63.251, P < 0.001; one-way ANOVA). D, Temporal expression profile of Pdcd1 mRNA in TAMs. Data were normalized by 18S rRNA levels. Values are the mean with SD (n ¼ 3). There was a significant time-dependent variation in Pdcd1 mRNA levels (F5,12 ¼ 4.167, P ¼ 0.020; one-way ANOVA). The horizontal bar at the bottom of each panel indicates light and dark cycles.

Article Snippet: Membranes were reacted with antibodies against DEC2 (12688–1-AP, Proteintech, Wuhan, China, RRID: AB_2065361), b-ACTIN conjugated with horseradish peroxidase (sc-47778, Santa Cruz Biotechnology, RRID:AB_2714189), p65 (ab16502, Abcam, Cambridge, Massachusetts), and p84 (10920–1- AP, Proteintech, RRID:AB_2202239).

Techniques: Expressing, Isolation

Journal: Molecular Cancer Research

Article Title: Diurnal Expression of PD-1 on Tumor-Associated Macrophages Underlies the Dosing Time-Dependent Antitumor Effects of the PD-1/PD-L1 Inhibitor BMS-1 in B16/BL6 Melanoma-Bearing Mice

doi: 10.1158/1541-7786.mcr-21-0786

Figure Lengend Snippet: Figure 2. DEC2 regulates the circadian expression of Pdcd1 mRNA in macrophages. A, Temporal mRNA expression profiles of Per2, Bmal1, and Pdcd1 in RAW264.7 cells, whose circadian clocks were synchronized by treatment with 100 nmol/L DEX for 2 hours. Nontreatment cells were set as a nonsynchronized control. Data were normalized by the levels of 18S rRNA and the mean of each group was set at 1.0. Values are the mean with SD (n ¼ 3). There were significant time-dependent variations in Per2, Bmal1, and Pdcd1 in DEX treatment group (F12, 26 ¼ 53.225, P < 0.001 for Per2; F12, 26 ¼ 12.609, P < 0.001 for Bmal1; F12, 26 ¼ 18.874, P < 0.001 for Pdcd1; one-way ANOVA). B, DEC2 negatively regulates the transcription of the Pdcd1 gene. RAW264.7 cells were cotransfected with Pdcd1(-2050/þ63)::Luc, and expression vectors for PER1, PER2, CRY1, DEC1, DEC2, CLOCK/BMAL1, NFIL3, DBP, RORa, and REV-ERBa. The values are the mean with SD (n ¼ 3). The value of empty vector (pcDNA3.1)-transfected RAW264.7 cells was set at 1.0. , P < 0.05; , P < 0.01; , P < 0.001; significant difference from empty vector (pcDNA3.1)-transfected groups (F10,33 ¼ 18.109, P < 0.001; ANOVA with the Tukey Kramer post hoc test). C, Temporal expression profiles of DEC2 protein in mock-transduced and Dec2 KD RAW264.7 cells, whose circadian clocks were synchronized by treatment with 100 nmol/L DEX. Data were normalized by the b-ACTIN levels. Values are the mean with SD (n ¼ 3). (F1, 28 ¼ 216.110, P < 0.01 for group; F6,28 ¼ 17.930, P < 0.01 for time point; F6,28 ¼ 12.966, P < 0.01 for time pointgroup; two-way ANOVA). D, Temporal expression profiles of Pdcd1 mRNA in mock-transduced and Dec2 KD RAW264.7 cells, whose circadian clocks were synchronized by treatment with 100 nmol/L DEX. Data were normalized by the levels of 18S rRNA. Values are the meanwith SD (n ¼ 3). (F1,28 ¼ 323.663, P < 0.01 for group; F6,28 ¼ 8.459, P ¼ 0.016 for time point; F6,28 ¼ 4.890, P < 0.01 for time pointgroup; two-way ANOVA).

Article Snippet: Membranes were reacted with antibodies against DEC2 (12688–1-AP, Proteintech, Wuhan, China, RRID: AB_2065361), b-ACTIN conjugated with horseradish peroxidase (sc-47778, Santa Cruz Biotechnology, RRID:AB_2714189), p65 (ab16502, Abcam, Cambridge, Massachusetts), and p84 (10920–1- AP, Proteintech, RRID:AB_2202239).

Techniques: Expressing, Control, Plasmid Preparation, Transfection

Journal: Molecular Cancer Research

Article Title: Diurnal Expression of PD-1 on Tumor-Associated Macrophages Underlies the Dosing Time-Dependent Antitumor Effects of the PD-1/PD-L1 Inhibitor BMS-1 in B16/BL6 Melanoma-Bearing Mice

doi: 10.1158/1541-7786.mcr-21-0786

Figure Lengend Snippet: Figure 3. Repression of NF-kB–mediated transactivation by DEC2 underlies the circadian expression of Pdcd1. A, Bioluminescence profiles driven by Pdcd1(-2050/þ63)::Luc-, Pdcd1(-1540/þ63)::Luc-, and Pdcd1(-913/þ63)::Luc-transfected NIH3T3 cells after treatment with 100 nmol/L DEX for 2 hours. The upper schematic diagrams show luciferase reporter constructs containing different lengths of the upstream region of the mouse Pdcd1 gene. Closed boxes indicate the sites homologous with clock gene response elements and the numbers of nucleotide residues indicate the distance from the transcription start site (þ1). Values are the mean with SD (n ¼ 6–8). B, Bioluminescence profiles driven by Pdcd1(-1540/þ63)::Luc in circadian clock-synchronized NIH3T3 cells transfected with Dec2-expressing vectors or control (pcDNA) vectors. C, Location of the NRE in the upstream region of the mouse Pdcd1 gene. D, Suppression of p65-mediated transactivation of the NRE::Luc by DEC2. RAW264.7 cells were cotransfected with NRE::Luc, and expression vectors for p65 and DEC2. Values are the mean with SD (n ¼ 3). The value of empty vector (pcDNA3.1)-transfected RAW264.7 cells was set at 1.0. , P < 0.01; significant difference between the two groups (F3,8 ¼ 237.051, P < 0.001; one-way ANOVA with Tukey Kramer post hoc test). E, Suppression of p65-mediated transactivation of the Pdcd1 (–1540/þ63)::Luc by DEC2. RAW264.7 cells were cotransfected with Pdcd1(–1540)::Luc, and expression vectors for p65 and DEC2. Values are the mean with SD (n ¼ 3). The value of empty vector (pcDNA3.1)-transfected RAW264.7 cells was set at 1.0. , P < 0.01; significant difference between the two groups (F3,8 ¼ 29.215, P < 0.001; one-way ANOVA with Tukey Kramer post hoc test). F, Suppression of LPS-induced nuclear translocation of p65 by DEC2. RAW264.7 cells were transfected with empty vector (pcDNA3.1) or Dec2-expressing vector and then treated with 1 mg/mL of LPS for 30 minutes. , P < 0.01; significant difference between the two groups (F3,8 ¼ 96.447, P < 0.001; one-way ANOVA with Tukey Kramer post hoc test).

Article Snippet: Membranes were reacted with antibodies against DEC2 (12688–1-AP, Proteintech, Wuhan, China, RRID: AB_2065361), b-ACTIN conjugated with horseradish peroxidase (sc-47778, Santa Cruz Biotechnology, RRID:AB_2714189), p65 (ab16502, Abcam, Cambridge, Massachusetts), and p84 (10920–1- AP, Proteintech, RRID:AB_2202239).

Techniques: Expressing, Transfection, Luciferase, Construct, Control, Plasmid Preparation, Translocation Assay

Journal: Molecular Cancer Research

Article Title: Diurnal Expression of PD-1 on Tumor-Associated Macrophages Underlies the Dosing Time-Dependent Antitumor Effects of the PD-1/PD-L1 Inhibitor BMS-1 in B16/BL6 Melanoma-Bearing Mice

doi: 10.1158/1541-7786.mcr-21-0786

Figure Lengend Snippet: Figure 4. Regulation of antitumor immunity of RAW264.7 macrophages by DEC2. (A and B) The viability of B16/BL6 melanoma (A) and mock-transduced or Dec2 KD RAW264.7 cells (B) after treatment with 10 mmol/L BMS-1 for 24 hours. C, BMS-1 increases the antitumor immunity of RAW264.7 cells under coculture conditions. B16/ BL6 melanoma was cocultured with mock-transduced or Dec2 KD RAW264.7 cells, and cells were treated with 10 mmol/L BMS-1 or vehicle (0.2% DMSO) for 24 hours. All experiments were conducted without synchronization of the circadian clock. Values are the mean with SD (n ¼ 6). , P < 0.01; significant difference between the two groups (F3,20 ¼ 419.160, P < 0.001; one-way ANOVA with Tukey Kramer post hoc test).

Article Snippet: Membranes were reacted with antibodies against DEC2 (12688–1-AP, Proteintech, Wuhan, China, RRID: AB_2065361), b-ACTIN conjugated with horseradish peroxidase (sc-47778, Santa Cruz Biotechnology, RRID:AB_2714189), p65 (ab16502, Abcam, Cambridge, Massachusetts), and p84 (10920–1- AP, Proteintech, RRID:AB_2202239).

Techniques:

Journal: Molecular Cancer Research

Article Title: Diurnal Expression of PD-1 on Tumor-Associated Macrophages Underlies the Dosing Time-Dependent Antitumor Effects of the PD-1/PD-L1 Inhibitor BMS-1 in B16/BL6 Melanoma-Bearing Mice

doi: 10.1158/1541-7786.mcr-21-0786

Figure Lengend Snippet: Figure 6. Schematic diagram underlying mechanism of the dosing time-dependent changes in the antitumor effects of PD-1/PD-L1 inhibitor BMS-1 in B16BL6 melanoma- implanted mice. The time-dependent repression of p65-mediated transactivation of Pdcd1 by DEC2 induces the circadian expression of PD-1 in TAMs. The antitumor efficacy of BMS-1 is enhanced by administering at the time of day when PD-1 expression is increased on TAMs.

Article Snippet: Membranes were reacted with antibodies against DEC2 (12688–1-AP, Proteintech, Wuhan, China, RRID: AB_2065361), b-ACTIN conjugated with horseradish peroxidase (sc-47778, Santa Cruz Biotechnology, RRID:AB_2714189), p65 (ab16502, Abcam, Cambridge, Massachusetts), and p84 (10920–1- AP, Proteintech, RRID:AB_2202239).

Techniques: Expressing

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: Expressing

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, Recombinant, Electron Microscopy, Staining, SYBR Green Assay, Software, Imaging