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: bioRxiv

Article Title: Matrix metalloproteinases proteolyze RAB proteins and contribute to cisplatin-induced ototoxicity

doi: 10.64898/2026.02.28.708770

Figure Lengend Snippet: Gelatin zymography (A, C, E) and corresponding quantitative analysis (B, D, F, G) of MMP-2 and MMP-9 activities in the conditioned media of HEI-OC1 cells following 6 h (A, B) and 48 h (C, D) of exposure to 20 μM cisplatin. Cytosolic MMP-2 and MMP-9 activities were measured following 4 h and 6 h of treatment with 20 μM cisplatin (E-G). Standard (Std) derives from conditioned medium from HT-1080 cells. H) mRNA expression of Ifit-1 was measured using RT-qPCR following a 24h exposure to 20 μM cisplatin in the presence or absence of ARP-100 (5 μM), compared to the untreated control (nil), and normalized to B2M expression. The data are represented as the mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test (* p < 0.05 and ** p <0.01).

Article Snippet: For overexpression studies, the pCMV3-C-FLAG vector encoding human MMP-2 ORF (HG10082-CF, Sino Biological) was transfected into HEI-OC1 using jetPRIME Reagent (Polyplus) according to the manufacturer’s protocol.

Techniques: Zymography, Expressing, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Matrix metalloproteinases proteolyze RAB proteins and contribute to cisplatin-induced ototoxicity

doi: 10.64898/2026.02.28.708770

Figure Lengend Snippet: mRNA expression of N-terminal truncated (NTT)- Mmp-2 (A), full-length (FL)- Mmp-2 (B), and Mmp-9 (C) was measured using RT-qPCR following exposure to 20 μM cisplatin for various timepoints, compared to the untreated control (0), and normalized to B2M expression. D) Immunofluorescence staining of HEI-OC1 cells treated with 20 μM cisplatin for 6 h or untreated cells. Staining was performed using an antibody specific to MMP-2, followed by detection with a secondary antibody conjugated to Alexa Fluor 647 (red). The zoomed-in boxes indicate nuclei with an increased red signal (magenta). The data are represented as the mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test (* p < 0.05).

Article Snippet: For overexpression studies, the pCMV3-C-FLAG vector encoding human MMP-2 ORF (HG10082-CF, Sino Biological) was transfected into HEI-OC1 using jetPRIME Reagent (Polyplus) according to the manufacturer’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Matrix metalloproteinases proteolyze RAB proteins and contribute to cisplatin-induced ototoxicity

doi: 10.64898/2026.02.28.708770

Figure Lengend Snippet: (A) IL-6 secretion in cells transfected with either an empty vector (EV) or an Mmp-2 -expressing vector and treated with 100 μM cisplatin or left untreated (nil) for 24 h. (B, C) IL-6 secretion in cells pre-treated for 2 h with the vehicle or 5 μM ARP-100 (B) or ONO-4817 (C), followed by treatment with 20 μM cisplatin for 24 h. (D) IL-6 secretion in cells transfected with non-targeting siRNA ( siNT ), Mmp-9 -targeting siRNA ( siMmp-9 ), and exposed to 20 μM cisplatin for 24 h. The data are represented as the mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test (* p < 0.05).

Article Snippet: For overexpression studies, the pCMV3-C-FLAG vector encoding human MMP-2 ORF (HG10082-CF, Sino Biological) was transfected into HEI-OC1 using jetPRIME Reagent (Polyplus) according to the manufacturer’s protocol.

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Matrix metalloproteinases proteolyze RAB proteins and contribute to cisplatin-induced ototoxicity

doi: 10.64898/2026.02.28.708770

Figure Lengend Snippet: A, B) Immunoblot of in vitro time course proteolysis of recombinant RAB9A at 37 °C with 100 ng MMP-2 (A) or for 30 minutes in the presence or absence of MMP-2-preferring inhibitors (B). C) In silico analysis of the top 10 MMP-2 cleavage sites in the structure of mouse RAB9A according to ProsperousPlus. D) Crystal structure of mouse RAB9A showing the predicted cleavage sites (green balls).

Article Snippet: For overexpression studies, the pCMV3-C-FLAG vector encoding human MMP-2 ORF (HG10082-CF, Sino Biological) was transfected into HEI-OC1 using jetPRIME Reagent (Polyplus) according to the manufacturer’s protocol.

Techniques: Western Blot, In Vitro, Recombinant, In Silico

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: RT-qPCR and WB of SIRT3 in scramble (SCR) and si SIRT3 conditions (A). Representative images of SCR and si SIRT3 in hNE cell cultures (B). SIRT3 silencing effects on SIRT family mRNA abundance in hNECs (C). RT-qPCR and WB of empty vector (EV) and SIRT3 overexpression (OE) conditions (D). Representative images of EV and SIRT3 OE in hNE cell cultures (E). SIRT3 OE effects on SIRT family mRNA abundance in hNECs (F). Data are presented as mean±SEM . *P<0.05 vs. control group (a.u: arbitrary units).

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Quantitative RT-PCR, Plasmid Preparation, Over Expression, Control

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: Overlap between down- and up-regulated DEGs (A) and DPPs (B) from SIRT3 silencing and overexpression conditions. Functional analysis of DEGs, DEPs and DPPs from SIRT3 silencing (si) (C) and overexpression (OE) (D).

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Over Expression, Functional Assay

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: Functional mapping of deregulated genes (DEGs), proteins (DEPs) and phosphoproteins (DPPs) upon SIRT3 silencing conditions in hNECs.

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: Functional mapping of deregulated genes (DEGs), proteins (DEPs) and phosphoproteins (DPPs) upon SIRT3 overexpressing conditions in hNECs.

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: Steady state protein levels of SIRT 1-3 were measured by Western blotting (WB) at 8-24-48 hours post-treatment (A). * p<0.05 respect to untreated-hNEC control cells. Overlap between down- and up-regulated DPPs from SIRT3 silencing and overexpression upon Aβ oligomer protein stimulation in hNECs (B). Selected differentially phosphorylated proteins affected by SIRT3 modulation in Aβ-treated cells (C). Functional analysis of DPPs from SIRT3 silencing (si) and overexpression (OE) (D).

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Western Blot, Control, Over Expression, Functional Assay

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: Predictive activation profile of pathways, biofunctions and upstream regulators at the level of SIRT3 silencing and/or overexpression upon Aβ oligomer considering APP as main hub. Positive z-scores indicate potential activated pathways, whereas negative z-scores refer to predicted inhibited pathways. Based on SIRT3 silencing and overexpression datasets (A). Activation prediction of significantly altered pathways and neuronal functions (B). Systems Biology analysis were performed through the Ingenuity Pathway Analysis software .

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Activation Assay, Over Expression, Software

Journal: Gene Therapy

Article Title: First use of gene therapy to treat growth hormone resistant dwarfism in a mouse model

doi: 10.1038/s41434-022-00313-w

Figure Lengend Snippet: a RT-PCR gel of mGHR mRNA expression. 18S rRNA housekeeping gene was used as loading control. b Western blot of mGHR protein expression. Actin housekeeping gene was used as loading control. c Immunofluorescence staining of mGHR expression, scale bars are 50 µm and nuclei were stained with DAPI. HepG2 cells were transfected with pAAV-HLP-mGHR consisted of hybrid liver-specific promoter (HLP) driving the expression of mGHR. Untransfected control and cells transfected with pAAV-HLP-Luc expressing luciferase gene were used as negative control. Analyses were performed 48 h post-transfection.

Article Snippet: The mGHR PCR fragment was amplified from cloning vector MG50043-M (Sino Biological, Beijing, China) containing mouse growth hormone receptor/GHR/GHBP transcript variant 1 gene ORF cDNA clone using forward primer with introducing NotI site: 5’-ATAAGAATGCGGCCGCACCATGGATCTTTGTCAGGTCTTC and reverse primer with introducing XhoI site: 5’-CCGCGCTCGAGCTACTGCATGATTTTGTTCAGTTG.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Negative Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Oligonucleotide primer sets for RT-qPCR.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Sequencing

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Exosomal transfer of NEAT1 to EC cells leads to increased expression of STAT3 and YKL-40. (A) RT-qPCR analysis of NEAT1 expression level in NFs, CAFs, normal endometrial epithelial cells, and EC cells. (B) HEC-1A and RL95-2 cells were cultured in control CM, CAFs-CM, or exosome-depleted CAFs-CM for 48 h, then NEAT1 level was assessed by RT-qPCR. HEC-1A and RL95-2 cells were maintained in control CM, CAFs-CM, or 20 μM GW4869-treated CAFs-CM for 48 h. (C) NEAT1 expression was determined by RT-qPCR. (D) The NEAT1 level was assessed by RT-qPCR. (E) STAT3, p-STAT3 and YKL-40 levels were assessed by Western blot. HEC-1A and RL95-2 cells were co-cultured with CAFs or GW4869-treated CAFs for 48 h. (F) Western blot analysis of STAT3, p-STAT3, and YKL-40 levels. (G) Western blot analysis of Rab27a protein level in CAFs infected with lentivirus expressing si-NC or si-Rab27a. (H) HEC-1A and RL95-2 cells were cultured in control CM, CAFs-CM, or Rab27a-silenced CAFs-CM for 48 h, then NEAT1 level was detected by RT-qPCR. (I) HEC-1A cells and RL95-2 were co-cultured with CAFs or Rab27a-silenced CAFs for 48 h, and NEAT1 level was detected by RT-qPCR. (J)&(K) Protein levels of STAT3, p-STAT3, and YKL-40 were assessed by Western blot. (L) RT-qPCR analysis of NEAT1 level in exosomes isolated from CAFs infected with lentivirus expressing sh-NEAT1 or NEAT1 gene. (M) HEC-1A and RL95-2 cells were incubated with exosomes derived from CAFs, NEAT1-overexpressed CAFs, or NEAT1-silenced CAFs for 48 h. RT-qPCR was performed to assess NEAT1 level. (N)&(O) STAT3 and YKL-40 expression was detected by RT-qPCR and Western blot assays. (P) The proliferation of HEC-1A and RL95-2 cells was detected by MTT assay. (Q) The growth of HEC-1A and RL95-2 cells was evaluated by colony formation assay. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Western Blot, Infection, Isolation, Incubation, Derivative Assay, MTT Assay, Colony Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Exosomal NEAT1 sponges miR-26a/b-5p to facilitate STAT3 and YKL-40 expression in EC cells. (A) RT-qPCR for miR-26a/b-5p level in exosomes derived from CAFs, NEAT1-overexpressed CAFs, or NEAT1-silenced CAFs. (B) HEC-1A and RL95-2 cells were co-cultured with NEAT1-overexpressed or silenced CAFs, and miR-26a/b-5p level was detected by RT-qPCR. (C) Expression of miR-26a/b-5p in HEC-1A and RL95-2 cells infected with lentivirus containing miR-26a/b-5p mimics or inhibitor was detected by RT-qPCR. (D) Expression of YKL-40 in HEC-1A and RL95-2 cells after overexpression or silencing of miR-26a/b-5p was assessed by Western blot. (E) The binding sites between NEAT1 and miR-26a/b-5p. Luciferase analysis (F) and RIP assay (G) was performed to determine interaction between NEAT1 and miR-26a/b-5p. The expression of STAT3 and YKL-40 in HEC-1A and RL95-2 cells received various treatments was assessed by RT-qPCR (H) and Western blot (I). All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001, ns = no significant.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Cell Culture, Infection, Over Expression, Western Blot, Binding Assay, Luciferase

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: MiR-26a/b-5p suppresses YKL-40 expression via targeting STAT3 in EC cells. (A) Two binding sites of miR-26a/b-5p to the 3’-UTR of STAT3. The interaction between STAT3 and miR-26a/b-5p was detected by luciferase analysis (B) and RIP assay (C). (D) The protein level of STAT3 in HEC-1A and RL95-2 cells after the transfection of STAT3 expression plasmid was assessed by Western blot. RT-qPCR (E) and Western blot (F) were used for determining YKL-40 and STAT3 expression in HEC-1A cells from various groups. (G) Three p-STAT3-binding sites (BS1, BS2, and BS3) at YKL-40 promoter were shown. (H) ChIP assay using anti-p-STAT3 antibody was used to verify the binding between p-STAT3 and the promoter of YKL-40 under exosome treatment. (I) The interaction between p-STAT3 and YKL-40 was assessed by the dual luciferase reporter assay. All data from three independent experiments were shown as mean ± SD (n = 6). **, P < 0.01; ***, P < 0.001, ns = no significant.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Reporter Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: CAFs-derived exosomal NEAT1 accelerates tumor growth in vivo via up-regulating YKL-40. Nude mice were subcutaneously injected with HEC-1A cells with or without CAFs. GW4869 (2 mg/kg) was intraperitoneally injected into the mice every other day. (A) The tumor growth curve was shown. (B) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (C) Immunohistochemical staining for Ki-67 expression in tumor tissues. (D) Western blot analysis of YKL-40 and STAT3 protein level in tumor tissues. Nude mice were subcutaneously injected with HEC-1A cells with or without CAFs that were stably transfected with sh-NC or sh-Rab27a. (E) The tumor growth curve was shown. (F) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (G) Immunohistochemical staining for Ki-67 expression in tumor tissues. (H) Western blot analysis of YKL-40 and STAT3 protein level in tumor tissues. HEC-1A cells with or without CAFs stably transfected with sh-NEAT1 or NEAT1 gene were subcutaneously injected into the nude mice. (I) The tumor growth curve was shown. (J) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (K) Immunohistochemical staining for Ki-67 expression in tumor tissues. (L) Western blot analysis of YKL-40 and STAT3 protein levels in tumor tissues. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Derivative Assay, In Vivo, Injection, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining, Western Blot, Stable Transfection, Transfection

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Overexpression of miR-26a/b-5p reverses exosomal NEAT1-mediated protumorigenic effect in vivo . HEC-1A cells stably transfected with miR-26a/b-5p mimics or NC mimics were mixed with or without CAFs stably expressing NC vector or NEAT1, which were subcutaneously injected into the nude mice. (A) The tumor growth curve was shown. (B) Immunohistochemical staining for Ki-67 expression in tumor tissues. (C) Western blot analysis of YKL-40 and STAT3 protein levels in tumor tissues. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Over Expression, In Vivo, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Injection, Immunohistochemical staining, Staining, Western Blot

Journal: NPJ Precision Oncology

Article Title: IL-11/IL-11R signal inhibition by 9MW3811 remodels immune tumor microenvironment and enhances anti-tumor efficacy of PD-1 blockade

doi: 10.1038/s41698-025-00913-w

Figure Lengend Snippet: A IL-11 is more overexpressed in CHOL, ESCA, HNSC, LUSC, LUAD, COAD, CESC, and BRCA (data from The Cancer Genome Atlas (TCGA) than normal tissues (data from the Genotype-Tissue Expression (GTEx). B Kaplan–Meier survival analysis of IL-11 mRNA levels for BRCA, CESC, CHOL, ACC, LUAD, PAAD, UCS, and COAD with datasets from TCGA and GTEx. C Relative contributions of IL-11 in different cancer types. The scores were evaluated by AIBERT®. D The impact of genetic loss of IL-11Rα on tumor growth was evaluated using MC38 cell line-derived tumor models with IL-11Rα knockout (IL-11Rα KO -MC38). MC38 or IL-11Rα KO -MC38 cells were inoculated subcutaneously into separate groups of C57BL/6J mice. Data are presented as mean ± SEM, n = 8. E The effects of genetic loss of IL-11Rα on tumor growth were assessed in Hepa1-6 tumor models using C57BL/6J wild-type mice and IL-11Rα knockout mice ( IL-11Rα KO -mice ). Hepa1-6 cells were inoculated subcutaneously into separate groups of C57BL/6 J wild-type or IL-11Rα KO mice, Data are presented as mean ± SEM, n = 6.

Article Snippet: IL-11Rα/STAT3-luc HEK293 reporter cell line was constructed by transfecting the expression vector containing human IL-11Rα full-length gene (HG10252-UT, Sino Biological) into STAT3-Luc/HEK293 cells which constitutively express human gp130 STAT3-luciferase (CBPB0002, Nanjing Cobioer).

Techniques: Expressing, Derivative Assay, Knock-Out

Journal: NPJ Precision Oncology

Article Title: IL-11/IL-11R signal inhibition by 9MW3811 remodels immune tumor microenvironment and enhances anti-tumor efficacy of PD-1 blockade

doi: 10.1038/s41698-025-00913-w

Figure Lengend Snippet: A Binding activity of 9MW3811 with recombinant IL-11 from different species. B Binding activity of 9MW3811 with human IL-11, compared with X203, a reported anti-IL-11 neutralizing antibody. C Blocking activity of 9MW3811 against the formation of IL-11/IL-11Rα/gp130 protein complex, compared with X203. D Schematic representation of JAK/STAT3 signaling downstream of IL-11. E JAK/STAT3 signaling transduction inhibitions by 9MW3811 and X203 were performed by luciferase reporter gene assay, using IL-11Rα/STAT3-luc HEK293 cell line. F The inhibition of pSTAT3 stimulated by human IL-11 in HGC-27 tumor cells was detected by immunoblotting assay using Simple Western-Jess. Data is shown as mean± SD.

Article Snippet: IL-11Rα/STAT3-luc HEK293 reporter cell line was constructed by transfecting the expression vector containing human IL-11Rα full-length gene (HG10252-UT, Sino Biological) into STAT3-Luc/HEK293 cells which constitutively express human gp130 STAT3-luciferase (CBPB0002, Nanjing Cobioer).

Techniques: Binding Assay, Activity Assay, Recombinant, Blocking Assay, Transduction, Luciferase, Reporter Gene Assay, Inhibition, Western Blot, Simple Western