Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A ) The predicted protein tertiary structure of CcTRPM . The conserved ankyrin repeat (ANK) domain was indicated in the N-terminal. The conserved six transmembrane domain of ion channels structure were shown as TM1-TM6. ( B ) The mRNA expression of CcTRPM in response to different temperatures of 25°C and 10°C by qRT-PCR. ( C ) Fluorescence detection of Fluo-4 AM after heterologous expression of CcTRPM in mammalian HEK293T cells in response to menthol treatment. The recombinant plasmid was generated by inserting the full ORF sequence of CcTRPM into pcDNA3.1(+)-mCherry plasmid. DMSO treatment and ionomycin treatment were used as negative control and positive control, separately. ( D ) Representative images of Ca 2+ imaging after heterologous expression of CcTRPM in mammalian HEK293T cells in response to different temperature treatment. “CcTRPM +10 °C” means the recombinant plasmid of CcTRPM with pcDNA3.1(+)-mCherry was treated with 10 °C. “CcTRPM +25 °C” denotes the pcDNA3.1(+)-mCherry plasmid with CcTRPM was treated with 25 °C. mCherry and Fluo-4 signal are shown in red and green, respectively. Scale bar is 20 μm. ( E ) The developmental expression pattern of CcTRPM for SF at mRNA level using qRT-PCR. 1st, 2nd, 3rd, 4th, and 5th are the nymphs at the first, second, third, fourth and fifth instar, respectively. A-1d, A-5d, and A-10d are the adults at 1 day, 5 days, and 10 days, separately. ( F ) Representative confocal images of CcTRPM in different developmental stages of SF using FISH. Scale bar is 0.5 mm. BF: the bright field. DAPI: the cell nuclei were stained with DAPI and visualized in blue. CcTRPM-Cy3: CcTRPM signal was labeled with Cy3 and visualized in red. Merge: merged imaging of BF, DAPI, and CcTRPM-Cy3 signals. ( G–H ) Comparison of the nymph cuticle ultrastructure, cuticle thickness, and cuticle chitin content of SF 1st instar treated with dsCcTRPM and dsEGFP at 15 days. ( I ) The transition percent of SF 1st instar nymphs treated with dsEGFP, DMSO, dsCcTRPM, CcTRPM antagonist and dsCcTRPM +menthol at 15 days under 10 °C. For RNAi experiments, summer-form 1st instar nymphs were fed with dsEGFP (500 ng/μL) or dsCcTRPM (500 ng/μL). To mimic RNAi effect, summer-form 1st instar nymphs were fed with 0.1% DMSO or CcTRPM antagonist (20 ng/μL). For the rescue experiment, summer-form 1st instar nymphs were fed with the mixture of dsCcTRPM (500 ng/μL) and menthol (1 mg/mL). Then, counted the number of summer-form and winter-form individuals and calculated the transition percent. ( J ) The phenotypes of SF 1st instar nymphs treated with dsEGFP, dsCcTRPM, CcTRPM antagonist and dsCcTRPM +menthol at 15 days under 10 °C. The data in 2B and 2E are shown as the mean ± SD with three independent biological replications of at least 30 nymphs for each biological replication. Scale bar is 0.5 mm. Data in 2 G and 2 H are presented as mean ± SD with three biological replications of three technical replicates for each biological replication. Data in 2I are presented as mean ± SD with nine biological replications. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by *** (p<0.001). Different letters above the bars indicated statistically significant differences (p<0.05), as determined by ANOVA followed with a Turkey’s HSD multiple comparison test in SPSS 20.0 software.

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Expressing, Quantitative RT-PCR, Fluorescence, Recombinant, Plasmid Preparation, Generated, Sequencing, Negative Control, Positive Control, Imaging, Staining, Labeling, Comparison, Software

Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A–B ) The chemical structure of menthol and TRPM antagonist. ( C–E ) Melting curve for qRT-PCR primers of CcTRPM , Ccβ-actin and CcEF-1 . ( F ) The mRNA expression of CcTRPM in response to different temperatures of 25°C and 10°C at 1 day and 2 days by qRT-PCR. ( G ) Effect of menthol treatment at different concentrations on the mRNA expression of CcTRPM under 10 °C condition. ( H ) Effect of TRPM antagonist treatment at different concentrations on the mRNA expression of CcTRPM under 10 °C condition. ( I ) Representative images of Ca 2+ imaging after heterologous expression of CcTRPM in mammalian HEK293T cells in response to menthol treatment. ‘CcTRPM +menthol’ means the recombinant plasmid of CcTRPM with pcDNA3.1(+)-mCherry was treated with menthol. “CcTRPM +DMSO” denotes the pcDNA3.1(+)-mCherry plasmid with CcTRPM was treated with DMSO. mCherry and Fluo-4 signal are shown in red and green, respectively. Scale bar is 20 μm. Data are shown as mean ± SD with three biological replications. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by *** (p<0.001). Different letters above the bars indicated statistically significant differences (p<0.05), as determined by ANOVA followed with a Turkey’s HSD multiple comparison test in SPSS 20.0 software.

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Quantitative RT-PCR, Expressing, Imaging, Recombinant, Plasmid Preparation, Comparison, Software

Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A ) The putative miR-252 binding sites in CcTRPM 3’UTR were predicted by miRanda and Targetscan. ( B ) The expression profiles of miR-252 in response to different temperatures of 25°C and 10°C by qRT-PCR. ( C ) In vitro validation of the target interactions between miR-252 and CcTRPM by dual luciferase reporter assays. ( D ) In vivo demonstration of miR-252 targeting CcTRPM by RNA-binding protein immunoprecipitation (RIP) assay. ( E ) Representative confocal images of miR-252 and CcTRPM in 1st, 3rd and 5th instar of SF. Scale bar is 0.5 mm. The signals of DAPI and CcTRPM -Cy3 are same as the above describing. miR-252-FAM: the miR-252 signal was labeled with FAM and visualized in green. Merge: merged imaging of co-localization of cell nucleus, CcTRPM and miR-252. ( F ) Effect of miR-252 agomir and antagomir treatment 3 days on the expression of CcTRPM at mRNA level. The data in 3B and 3 F are shown as the mean ± SD with three independent biological replications of at least 30 nymphs for each biological replication. Data in 3 C are presented as mean ± SD with nine biological replications. Data in 3D are presented as mean ± SD with two biological replications of three technical replicates for each biological replication. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by *** (p<0.001). Different letters above the bars indicated statistically significant differences (p<0.05), as determined by ANOVA followed with a Turkey’s HSD multiple comparison test in SPSS 20.0 software.

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, In Vitro, Biomarker Discovery, Luciferase, In Vivo, RNA Binding Assay, Immunoprecipitation, Labeling, Imaging, Comparison, Software

Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A ) Effect of miR-252 agomir treatment on the mRNA expression of miR-252 after 3 days and 6 days under 10 °C. agomir-NC treatment was used as the control. ( B ) The mRNA expression of miR-252 after dsCcTRPM treatment 3 days and 6 days compare with the dsEGFP treatment under 10 °C. ( C–F ) Comparison of the nymph cuticle thickness, cuticle ultrastructure, cuticle chitin staining with WGA-FITC, and chitin content of SF 1st instar after treatment with agomir-NC and agomir-252 at 15 days. Scale bar in (D) is 1μm and in (E) is 100 μm. The two-way arrow indicated the cuticle thickness. The DAPI and WAG-FITC signals were same as the above describing. ( G ) The transition percent of SF 1st instar nymphs treated with agomir-NC, agomir-252, dsEGFP, dsCcTRPM, and dsCcTRPM +antagomir-252 at 15 day under 10 °C. ( H ) The phenotypes of SF 1st instar nymphs treated with agomir-NC, agomir-252, dsCcTRPM, and dsCcTRPM +antagomir-252 at 15 day under 10 °C. Scale bar is 0.5 mm. The data in 4A and 4B are shown as the mean ± SD with three independent biological replications of at least 30 nymphs for each biological replication. Data in 4C and 4F are presented as mean ± SD with three biological replications of three technical replications for each biological replication. Data in 4G are presented as mean ± SD with nine biological replications. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by *** (p<0.001). Different letters above the bars indicated statistically significant differences (p<0.05), as determined by ANOVA followed with a Turkey’s HSD multiple comparison test in SPSS 20.0 software.

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Expressing, Control, Comparison, Staining, Software

Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A–B ) Melting curve for qRT-PCR primers of U6 and miR-252. ( C ) Effect of agomir-252 treatment 2 day and 4 day on the mRNA expression of CcTRPM under 25 °C condition. ( D ) The phenotype of SF 1st instar nymphs treated with agomir-NC and agomir-252 at 10 day at 25 °C. Scale bar is 0.5 mm. Data are shown as the mean ± SD with three independent biological replications of at least 30 nymphs for each biological replication. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by *** (p<0.001).

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

doi: 10.7554/eLife.88744

Figure Lengend Snippet: ( A ) Diagram of de novo biosynthesis of insect chitin. Tre: trehalase. HK: hexokinase. G6PI: glucose-6-phosphate isomerase. GFAT: fructose-6-phosphate aminotransferase. GNA: glucosamine-6-phosphate acetyltransferase. AGM: N-acetylglucosamine phosphate mutase. UAP: UDP- N-acetylglucosamine pyrophosphorylase. CHS: chitin synthase. ( B ) A heat map was constructed from the expression levels of chitin biosynthesis enzyme transcripts in C. chinensis after 25°C and 10°C treatment at 3 d, 6 d, and 10 d. ( C ) Effect of RNAi knockdown of 14 transcripts covering all the enzymes in the chitin biosynthesis pathway on the transition percent of SF 1st instar nymphs under 10 °C. ( D–F ) Comparison of the nymph cuticle thickness, cuticle chitin content, and cuticle chitin staining with WGA-FITC of SF 1st instar after treatment with dsEGFP, dsCcTre1, and dsCcCHS1 at 15 d. The WAG-FITC signal was same as the above describing. Scale bar in (F) is 100 μm and 0.5 mm, respectively. ( G–H ) Effect of dsCcTRPM and agomir-252 treatments on the mRNA expression of CcTre1 at 3 day and 6 day under 10 °C. ( I–J ) Effect of dsCcTRPM and agomir-252 treatments on the mRNA expression of CcCHS1 at 3 day and 6 day under 10 °C. The data in ( C ) are shown as the mean ± SD with six independent biological replications of at least 30 nymphs for each biological replication. Data in ( D and E ) are presented as mean ± SD with three biological replications of three technical replications for each biological replication. Data in ( G-J ) are presented as mean ± SD with three biological replications. Statistically significant differences were determined with the pair-wise Student’s t -test, and significance levels were denoted by * (p<0.05), ** (p<0.01), and *** (p<0.001).

Article Snippet: We therefore attempted to identify chemical compounds that either activate or inhibit CcTRPM . qRT-PCR results revealed that menthol treatment with all three concentrations significantly increased the mRNA expression of CcTRPM and antagonist (HY-112430, MedChemExpress, Shanghai, China) treatment with all three concentrations markedly reduced CcTRPM transcription ( ).

Techniques: Construct, Expressing, Knockdown, Comparison, Staining

Journal: iScience

Article Title: Development of the FGFR4-ADC bearing the ferroptosis inducer sulfasalazine for hepatocellular carcinoma

doi: 10.1016/j.isci.2025.113718

Figure Lengend Snippet: FGFR4 is up-regulated in HCC and positively correlated with poorer prognosis (A) The mRNA expression levels of FGFR4 in different types of tumors in the HPA database. (B) The mRNA expression levels of FGFR4 in different liver cancer cell lines in the HPA database. (C) The protein expression levels of FGFR4 in HCC cell lines (Huh-7, Hep 3B, Hep-G2, and SK-HEP-1) was analyzed by western blot. (D) The mRNA expression of FGFR4 in HCC tumors compared to matched normal tissues using the GEPIA2 and UALCAN databases. ∗∗∗∗, p < 0.0001. (E–H) Kaplan-Meier analysis of overall survival, disease-free survival, and progression-free survival of patients with HCC with FGFR4 different expression was performed using KM plotter, OncoLnc, and GEPIA2 databases.

Article Snippet: An analysis was conducted on the publicly accessible mRNA database, the Human Protein Atlas (HPA) ( www.proteinatlas.org ), to identify antigens that could potentially serve as suitable targets for the development of antibody-based therapeutics.

Techniques: Expressing, Western Blot