shrna plasmid with pspax2  (Addgene inc)

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: NRDC is a direct target of miR-136-3p in human myotubes. Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24). * p < 0.05, ** p < 0.005. GSE = gene set enrichment; HEK293 = human embryonic kidney; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; nTPM = normalized transcripts per million; si NRDC = small interfering RNA of NRDC ; UTR = untranslated region.

Article Snippet: MiR-136-3p was labeled with Cy3 using Silencer small interfering RNA (siRNA) Labeling Kit with Cy3 Dye (Thermo Fisher Scientific) and loaded into exosome-enriched EVs with Exo-Fect siRNA/miRNA Transfection Reagent (System Biosciences, Palo Alto, CA, USA).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative Proteomics, Negative Control, Small Interfering RNA

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: Cellular metabolism in human myotubes after miR-136-3p transfection or NRDC silencing. Mitochondrial respiration in miR-136-3p-transfected or NRDC- silenced human myotubes was monitored using the Mitochondrial Stress Test. (A) OCR and (B) ECAR were measured using the Seahorse XFe24 Extracellular Flux Analyzer. The trace shows representative data. (C) Quantification of the mitochondrial respiration data for basal respiration, maximal respiration, ATP production, and spare respiratory capacity obtained from 3 independent experiments. Human myotubes were transfected with miR-136-3p or siRNA against NRDC before determination of (D) uptake of radiolabeled glucose, (E) rates of radiolabeled glucose oxidation, (F) conversion of radiolabeled glucose into glycogen, (G) rate of radiolabeled palmitic acid oxidation, (H) protein synthesis as assessed by incorporation of puromycin, and (I) lactate release into the media. Results are expressed as mean ± standard error of the mean. * p < 0.05, ** p < 0.005 vs. control cells. ECAR = extracellular acidification rate; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; ns = no significance; OCR = oxygen consumption rate; OigoA = oligomycin A; Rot/AA = rotenone and antimycin A; si NRDC = small interfering RNA of NRDC; siRNA = small interfering RNA; scr = negative control for small interfering RNA.

Article Snippet: MiR-136-3p was labeled with Cy3 using Silencer small interfering RNA (siRNA) Labeling Kit with Cy3 Dye (Thermo Fisher Scientific) and loaded into exosome-enriched EVs with Exo-Fect siRNA/miRNA Transfection Reagent (System Biosciences, Palo Alto, CA, USA).

Techniques: Transfection, Control, Negative Control, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with siRNA targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Expressing, Methylation, MSP Assay, Over Expression, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Immunoprecipitation, Western Blot, Control, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: Inhibition of DNMT1 abrogates LINC00184-induced PI3K/AKT pathway activation and functional phenotypes. (A) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in KYSE-150 cells following overexpression of LINC00184 and treatment with 5-AZA. (B) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (A). (C) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (A). (D) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in TE-1 cells following overexpression of LINC00184 and treatment with 5-AZA. (E) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (D). (F) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (D). (G) Western blotting analysis of p-AKT and total AKT levels in esophageal squamous cell carcinoma cells under the following conditions: Control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. (H) Quantitative analysis of the pAKT/AKT protein expression level corresponding to the immunoblots presented in (G). Data are presented as mean ± SEM (n=3). Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Inhibition, Activation Assay, Functional Assay, Western Blot, Expressing, Over Expression, Derivative Assay, Control, Negative Control, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: DNMT1 inhibition reverses LINC00184-induced malignant phenotypes in ESCC cells. (A) Cell viability of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (B) Representative images of migrated KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (C) Representative flow cytometry dot plots showing the apoptosis rate of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (D) Statistical counting of migrated KYSE-150 cells corresponding to (B) (n=3). (E) Quantitative analysis of the apoptosis rate of KYSE-150 cells corresponding to (C) (n=3). (F) Cell viability of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (G) Representative images of migrated TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (H) Representative flow cytometry dot plots showing the apoptosis rate of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (I) Statistical counting of migrated TE-1 cells corresponding to (G) (n=3). (J) Quantitative analysis of the apoptosis rate of TE-1 cells corresponding to (H) (n=3). (K) Cell viability detected via Cell Counting Kit-8 assay in ESCC cells under the conditions of control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. Data are presented as mean ± SEM (n=3), Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine; ESCC, esophageal squamous cell carcinoma.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Inhibition, Over Expression, Flow Cytometry, Cell Counting, Control, Negative Control, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with siRNA targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.

Article Snippet: In total, three siRNA duplexes targeting distinct regions of LINC00184 (sequences listed in ) were chemically synthesized by Sangon Biotech Co., Ltd. KYSE-150 and TE-1 cells were transfected in 6-well plates using Lipofectamine ® RNAiMAX (Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Methylation, MSP Assay, Over Expression, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Immunoprecipitation, Western Blot, Control, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: Inhibition of DNMT1 abrogates LINC00184-induced PI3K/AKT pathway activation and functional phenotypes. (A) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in KYSE-150 cells following overexpression of LINC00184 and treatment with 5-AZA. (B) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (A). (C) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (A). (D) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in TE-1 cells following overexpression of LINC00184 and treatment with 5-AZA. (E) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (D). (F) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (D). (G) Western blotting analysis of p-AKT and total AKT levels in esophageal squamous cell carcinoma cells under the following conditions: Control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. (H) Quantitative analysis of the pAKT/AKT protein expression level corresponding to the immunoblots presented in (G). Data are presented as mean ± SEM (n=3). Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine.

Article Snippet: In total, three siRNA duplexes targeting distinct regions of LINC00184 (sequences listed in ) were chemically synthesized by Sangon Biotech Co., Ltd. KYSE-150 and TE-1 cells were transfected in 6-well plates using Lipofectamine ® RNAiMAX (Thermo Fisher Scientific, Inc.).

Techniques: Inhibition, Activation Assay, Functional Assay, Western Blot, Expressing, Over Expression, Derivative Assay, Control, Negative Control, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: DNMT1 inhibition reverses LINC00184-induced malignant phenotypes in ESCC cells. (A) Cell viability of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (B) Representative images of migrated KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (C) Representative flow cytometry dot plots showing the apoptosis rate of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (D) Statistical counting of migrated KYSE-150 cells corresponding to (B) (n=3). (E) Quantitative analysis of the apoptosis rate of KYSE-150 cells corresponding to (C) (n=3). (F) Cell viability of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (G) Representative images of migrated TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (H) Representative flow cytometry dot plots showing the apoptosis rate of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (I) Statistical counting of migrated TE-1 cells corresponding to (G) (n=3). (J) Quantitative analysis of the apoptosis rate of TE-1 cells corresponding to (H) (n=3). (K) Cell viability detected via Cell Counting Kit-8 assay in ESCC cells under the conditions of control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. Data are presented as mean ± SEM (n=3), Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine; ESCC, esophageal squamous cell carcinoma.

Article Snippet: In total, three siRNA duplexes targeting distinct regions of LINC00184 (sequences listed in ) were chemically synthesized by Sangon Biotech Co., Ltd. KYSE-150 and TE-1 cells were transfected in 6-well plates using Lipofectamine ® RNAiMAX (Thermo Fisher Scientific, Inc.).

Techniques: Inhibition, Over Expression, Flow Cytometry, Cell Counting, Control, Negative Control, Small Interfering RNA

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: NSUN5 regulates the m5C modification and expression of its downstream target gene GLUT1. (A) Dot blot assay illustrating global m 5 C modification levels of total RNA in shNC or shNSUN5 A549/DDP cells. (B) Distribution profile of m 5 C modifications across diverse RNA regions (CDS, downstream, exon, intron, upstream, 3′UTR, and 5′UTR) from RNA Bis-seq in shNC- and shNSUN5-transfected A549/DDP cells. (C) Line chart depicting m 5 C site distribution by methylation level after NSUN5 knockdown. (D) Expression of differentially expressed genes (DEGs) from RNA-seq analysis of shNC- vs. shNSUN5-transfected A549/DDP cells. (E) Enriched pathways of those DEGs (D) in the RNA-seq. (F) Venn diagram of significantly m 5 C-modified genes (BiS-seq) and DEGs (RNA-seq). (G) Integrated volcano plot showing methylation (BiS-seq) and expression (RNA-seq) changes for 149 overlapping genes. GLUT1 exhibited the most pronounced methylation decrease in hypo-down group. (H) Correlation between NSUN5 and GLUT1 mRNA expression in TCGA-LUAD cohort. (I) IHC of NSUN5 and GLUT1 in serial sections from the same LUAD tumor tissue sample (left). Frequency of GLUT1 overexpression stratified by high/low NSUN5 expression. Scale bars (the upper panel), 200 μm. Scale bars (the lower panel), 50 μm. (J) Representative immunofluorescence staining showing the subcellular localization of GLUT1 (red) in shNC or shNSUN5 A549/DDP cells. Nuclei were stained with DAPI (blue). Scale bars, 15 μm. (K) Protein expression of GLUT1 in shNC and NSUN5-knockdown cells was assessed by Western blot assays. (L) m 5 C-MeRIP-qPCR analysis showing m 5 C modification of GLUT1 mRNA in shNC- or shNSUN5-transfected A549/DDP cells. (M) GLUT1 mRNA stability after actinomycin D (4 μg/mL) treatment. Half-life calculated from decay curves. (N) Western blot assays evaluating relative GLUT1 protein expression in NSUN5-overexpressing vs. control cells. (O) m 5 C-MeRIP-qPCR quantifying m 5 C modification levels of GLUT1 mRNA in NSUN5-overexpressing vs. control cells. (P) Actinomycin D assay determining GLUT1 mRNA half-life in NSUN5-overexpressing vs. control cells. Rep: Repeat. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (L, O), Pearson correlation test (H) or Chi-square test (I). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: Modification, Expressing, Dot Blot, Transfection, Methylation, Knockdown, RNA Sequencing, Over Expression, Immunofluorescence, Staining, Western Blot, Control

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: NSUN5 confers cisplatin resistance in a GLUT1-dependent manner in vitro and in vivo . (A) Effect of GLUT1 knockdown on cisplatin sensitivity in NSUN5-overexpressing cells. Cellular viability and cisplatin IC 50 values were determined by CCK-8 assay in NSUN5-overexpressing A549 cells following GLUT1 knockdown. (B) Effect of GLUT1 knockdown on cisplatin-induced apoptosis in NSUN5-overexpressing cells. Apoptosis was assessed by flow cytometry in NSUN5-upregulated A549 cells after GLUT1 knockdown and cisplatin exposure. (C) Western blot analysis of indicated proteins in NSUN5-overexpressing A549 (left panel) and PC9 (right panel) cells, with or without cisplatin exposure and with or without GLUT1 knockdown. (D) Representative comet assay images (left panel) and quantitative tail moment analysis (right panel) in NSUN5-overexpressing A549 cells following GLUT1 knockdown. (E) Immunofluorescence showing nuclear γ-H2AX foci density in designated treatment groups. Scale bars, 10 μm. (F) Bioluminescence images of xenograft tumors across groups. (G) Tumor volume measurements in nude mice under indicated conditions. (H) Terminal tumor weights across groups. (I) H&E staining and IHC for NSUN5, GLUT1, p -RPA2, γ-H2AX, and Cleaved Caspase 3 (Cleaved C3) in mice tumor sections. Scale bars (the upper panel), 200 μm. Scale bars (the lower panel), 50 μm. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (A, D, E, H). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: In Vitro, In Vivo, Knockdown, CCK-8 Assay, Flow Cytometry, Western Blot, Single Cell Gel Electrophoresis, Immunofluorescence, Staining

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: Cisplatin-induced ROS enhances methyltransferases activity of NSUN5 to promote m 5 C modification of GLUT1 mRNA. (A, B) NSUN5-bound m 5 C RNA detection by Co-IP. Western blot revealed m 5 C-modified RNA bound by HA-NSUN5 treated with cisplatin or Tempol. (C) Three-step catalytic mechanism of NSUN5-mediated m 5 C methylation. First, deprotonated Cys359 (motif VI, purple) initiated nucleophilic attack on cytosine C6, forming a covalent S-thioester intermediate (II) that polarizes C5. Second, Cys308 (motif IV, orange) abstractd the C5 proton, enabling methyl transfer from SAM to generate methylated intermediate (III). Finally, general base-catalyzed β-elimination released m 5 C-modified RNA and regenerates the enzyme. Top: Amino acid sequence alignment of regions forming the active sites of m 5 C methyltransferases NSUN5; The conserved motifs of NSUN5 (IV and VI) were boxed. Bottom: Reaction pathway of m 5 C formation. (D) Schematic of single-site (NSUN5 C308A , NSUN5 C359A ) and double mutant (NSUN5 DM ) constructs. Domains: N-terminal globular (green), RNA methyltransferase (blue), C-terminal (grey). Catalytic cysteines (C308/C359, orange) and SAM binding site (pink) were shown. Amino acid positions were numbered from the N-terminus. (E) Western blot revealed m 5 C-modified RNA bound by wild-type or mutant HA-NSUN5 treated with cisplatin or Tempol. (F) RNA pull-down assay coupled with Western blot validated NSUN5 as a binding protein for GLUT1 mRNA in resistant cells. (G) RNA immunoprecipitation (left panel) and agarose gel electrophoresis (right panel) assays confirmed direct binding between NSUN5 protein and GLUT1 mRNA in A549/DDP cells. (H) Western blot of GLUT1 expression after overexpression of NSUN5 WT , NSUN5 C308A , or NSUN5 C359A in A549 cells under cisplatin treatment. (I) RIP assay comparing the binding ability of NSUN5 with GLUT1 mRNA in overexpressed NSUN5 WT , NSUN5 C308A or NSUN5 C359A cells when treated with cisplatin or Tempol. (J) m 5 C-MeRIP-qPCR analysis of GLUT1 mRNA m 5 C modification levels in cells transfected with wild-type or single-point mutation constructs, following cisplatin or Tempol treatment. (K) GLUT1 mRNA half-life measured by actinomycin D assay after NSUN5 WT versus NSUN5 DM overexpression in A549 cells after cisplatin exposure. (L) Luciferase activity of wild-type and m 5 C-site-mutated GLUT1 reporters in A549 cells overexpressing NSUN5 WT or NSUN5 DM . Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (G, I, J, L). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: Activity Assay, Modification, RNA Detection, Co-Immunoprecipitation Assay, Western Blot, Methylation, Sequencing, Mutagenesis, Construct, Binding Assay, Pull Down Assay, RNA Immunoprecipitation, Agarose Gel Electrophoresis, Expressing, Over Expression, Transfection, Luciferase

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: NSUN5-catalyzed m 5 C modification of GLUT1 mRNA maintains its YBX1-mediated stability. (A) Silver staining of whole-cell extract, biotin-NC pull-down (Bio-NC), and biotin-GLUT1 mRNA (Bio-GLUT1) pull-down proteins from A549/DDP cells (left panel). HPLC-MS/MS results showing the sequence HT score and relative abundance of YBX1 (right panel). (B) Correlation between YBX1 and GLUT1 mRNA expression in TCGA-LUAD cohort. (C) IHC staining of serial sections from the same LUAD patients showing co-expression of YBX1 and GLUT1. Scale bars (the upper panel), 200 μm. Scale bars (the lower panel), 50 μm. (D, E) GLUT1 expression at mRNA and protein levels following YBX1 depletion (shRNA #1/#2) in cisplatin resistant cells. (F) GLUT1 mRNA half-life determined by actinomycin D chase assay after YBX1 knockdown in A549/DDP cells. (G, H) GLUT1 mRNA (G, qPCR) and protein (H, Western blot) expression upon YBX1 overexpression in cisplatin sensitive LUAD cells. (I) GLUT1 mRNA half-life was measured by actinomycin D assay after YBX1 overexpression. (J) RIP assay showing enrichment of GLUT1 mRNA by the YBX1 antibody compared with the negative control IgG. (K) RNA-pulldown assay demonstrating direct binding between GLUT1 mRNA and YBX1. (L) Western blotting showed that YBX1 depletion reversed the increase in GLUT1 protein levels induced by NSUN5 overexpression upon cisplatin exposure. (M) RIP analysis evaluating YBX1 binding to GLUT1 mRNA in A549 cells overexpressing NSUN5 WT or NSUN5 DM with cisplatin treatment. (N) Dual-luciferase reporter assay measuring YBX1-mediated activity of GLUT1-WT and GLUT1-MUT reporters. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (D, G, J, M, N), Pearson correlation test (B) or Chi-square test (C). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: Modification, Silver Staining, Tandem Mass Spectroscopy, Sequencing, Expressing, Immunohistochemistry, shRNA, Knockdown, Western Blot, Over Expression, Negative Control, Binding Assay, Luciferase, Reporter Assay, Activity Assay

Journal: Redox Biology

Article Title: Oxidative stress-driven m 5 C methylation by NSUN5 confers cisplatin resistance in lung adenocarcinoma through promoting glycolysis

doi: 10.1016/j.redox.2026.104193

Figure Lengend Snippet: NSUN5 promotes glycolysis and HR through GLUT1. (A) The glucose uptake was measured in NSUN5-overexpressing A549 cells with shNC or shGLUT1 transfection by fluorescently labeled glucose analogue 2-NBDG. The nucleus (blue) was stained with Hoechst. Scale bars, 100 μm. (B) Glycolytic flux analysis by extracellular acidification rate (ECAR). Real-time ECAR tracing in A549 cells sequentially treated with glucose, oligomycin (oligo), and 2-DG across experimental groups (left panel). Quantification of glycolytic parameters, including the basal glycolytic rate, maximal glycolytic capacity, and spare glycolytic capacity (right panel). (C) Mitochondrial respiration analysis by oxygen consumption rate (OCR). Real-time OCR tracing in A549 cells sequentially treated with oligomycin, FCCP, and rotenone/antimycin A across groups (left panel). Quantification of mitochondrial parameters, including basal respiration, ATP production, maximal respiration, and spare respiratory capacity (right panel). (D) Relative lactate production in designated A549 cell groups. (E) Schematic representation of the HR reporter. (F) The HR levels of the indicated HEK293T cells were detected by flow cytometry. (G-J) Representative immunofluorescence images of MRE11 (G), p -RPA2 (H), BrdU (I), and RAD51 (J) foci in A549 cells under indicated treatments. Scale bars, 10 μm. Data were representative of at least three independent experiments and presented as mean (SD). Statistical significance was determined using Student's t-test (B-D, F-J), ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n.s, not significant.

Article Snippet: Short hairpin RNA (shRNA) oligonucleotides targeting NSUN5, YBX1, and GLUT1, as well as lentiviruses encoding NSUN5 and YBX1 overexpression constructs, were purchased from GeneChem (Shanghai, China).

Techniques: Transfection, Labeling, Staining, Flow Cytometry, Immunofluorescence

Journal: Bioactive Materials

Article Title: Spatiotemporal reprogramming of cardiac lipid metabolism by platelet-engineered RNA therapy epigenetically modulate heart repair and regeneration

doi: 10.1016/j.bioactmat.2026.02.009

Figure Lengend Snippet: Preparation, physicochemical characterization, and in vitro bioactivity of siCD36@MSNs. (A) Representative TEM images showing uniform spherical MSNs and an increase in particle size following siCD36 loading. Scale bar = 200 nm. (B-C) Dynamic light scattering (DLS) analysis of hydrodynamic size distributions of MSNs and siCD36@MSNs, confirming size increase upon siRNA incorporation. n = 3. (D-E) Zeta potential measurements showing a surface charge shift from −17.0 ± 0.1 mV (MSNs) to +25.8 ± 0.1 mV (siCD36@MSNs), consistent with Ca 2+ -mediated electrostatic complexation of siRNA. n = 3. (F) In vitro siRNA release profile of siCD36@MSNs at 37 °C in PBS, demonstrating a burst-dominated release during early time points followed by residual release phase extending to 24 h n = 3. (G-H) Western blot analysis and quantification showing efficient and specific knockdown of CD36 in neonatal mouse cardiomyocytes (NMCMs) treated with siCD36@MSNs, whereas scrambled siRNA@MSNs exhibited no significant effect. n = 3. (I-J) Immunofluorescence staining confirming reduced CD36 expression in NMCMs following siCD36@MSNs treatment. Scale bar = 20 μm. n = 3. (K-L) BODIPY probing and quantitative analysis showing reduced fatty acid uptake and lower intracellular lipid content in CMs treated with siCD36@MSNs, compared with control and scrambled-siRNA@MSNs which show no significant differences. Scale bar = 20 μm. n = 3. (M − N) Western blot analysis and quantification demonstrating upregulation of Cyclin E1 following CD36 knockdown, indicating reactivation of cell-cycle–associated signaling. n = 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns = non-significant.

Article Snippet: Three siRNA duplexes targeting different regions of the murine CD36 gene were synthesized (Synthace),And the sequences were provided in the supply.

Techniques: In Vitro, Zeta Potential Analyzer, Western Blot, Knockdown, Immunofluorescence, Staining, Expressing, Control

Journal: Bioactive Materials

Article Title: Spatiotemporal reprogramming of cardiac lipid metabolism by platelet-engineered RNA therapy epigenetically modulate heart repair and regeneration

doi: 10.1016/j.bioactmat.2026.02.009

Figure Lengend Snippet: CD36 silencing induces comprehensive reprogramming of the cardiomyocyte lipidome and associated metabolic pathways. Untargeted metabolomics analysis was performed on neonatal mouse cardiomyocytes (NMCMs) treated with siCD36 or control siRNA. n = 3. (A) Chemical classification of 1793 detected metabolites, with lipids and lipid-like molecules representing 34.46% of the total metabolome. (B) Principal component analysis (PCA) showing clear separation between control and siCD36-treated samples along PC1 (31.6% variance), indicating distinct global metabolic profiles. (C) Volcano plot identifying 272 significantly altered metabolites (VIP >0.5, P < 0.05) in siCD36-treated cardiomyocytes, with 175 upregulated (pink) and 97 downregulated (green) species relative to control. (D) Chemical class distribution of the 272 differential metabolites, demonstrating that lipids and lipid-like molecules constitute the largest category of altered species (37.93%). (E) Metabolic pathway enrichment analysis (pathway impact vs. statistical significance) highlighting primary bile acid biosynthesis, steroid biosynthesis, sphingolipid metabolism, and glutathione metabolism as the most significantly perturbed pathways. (F) Subclass distribution of differential lipid metabolites, showing glycerophospholipids (42.42%) as the predominant altered lipid category, followed by prenol lipids, fatty acyls, sphingolipids, and steroid derivatives. (G) Volcano plot of lipid-specific differential metabolites between control and siCD36 groups (VIP >0.5). (H) Hierarchical clustering heatmap of differentially abundant lipid species across individual replicates, revealing coordinated reprogramming of glycerophospholipid, prenol lipid, sphingolipid, and steroid metabolic networks. (I) Normalized abundance scores across lipid subclasses, demonstrating widespread remodeling with enrichment in prenol lipids and glycerophospholipids, and depletion in sphingolipids following CD36 knockdown.

Article Snippet: Three siRNA duplexes targeting different regions of the murine CD36 gene were synthesized (Synthace),And the sequences were provided in the supply.

Techniques: Control, Knockdown

Journal: Bioactive Materials

Article Title: Spatiotemporal reprogramming of cardiac lipid metabolism by platelet-engineered RNA therapy epigenetically modulate heart repair and regeneration

doi: 10.1016/j.bioactmat.2026.02.009

Figure Lengend Snippet: Platelet-mediated encapsulation enables thrombin-responsive siRNA release and preserves CD36 silencing activity in CMs. (A) Transmission electron microscopy (TEM) images showing uptake of siCD36@MSNs into platelet OCS invaginations without disruption of platelet ultrastructure. Scale bar = 400 nm (B) Confocal microscopy showing co-localization of DiO-labeled platelets (green) and Cy5-labeled siCD36@MSNs (magenta), confirming stable platelet encapsulation. Scale bar = 0.5 μm. (C) In vitro release profile of Cy5-siCD36 from platelet-encapsulated siCD36@MSNs (siCD36@MSNs@Plt) in the presence of thrombin (100 U mL −1 ) at 37 °C, showing a burst-dominated early release followed by a slower residual release phase. Platelets without siRNA loading served as controls. n = 3. (D) Dose-dependent siRNA release from siCD36@MSNs@Plt following 12 h thrombin stimulation (0–200 U mL −1 ). n = 3. (E) Schematic illustration of the Transwell system used to evaluate thrombin-triggered release and downstream delivery of siCD36@MSNs@Plt to neonatal mouse cardiomyocytes (NMCMs). (F) Confocal imaging demonstrating enhanced cellular uptake of siCD36@MSNs released from platelets upon thrombin stimulation (100 U mL −1 ) compared with unstimulated conditions. Scale bar = 5 μm. (G-H) Western blot analysis and quantification showing enhanced CD36 knockdown in NMCMs treated with thrombin-activated siCD36@MSNs@Plt. n = 3. (I-J) Immunofluorescence analysis confirming reduced CD36 expression under the same conditions. Scale bar = 20 μm. n = 3. (K-L) BODIPY staining and quantification showing reduced fatty acid uptake and intracellular lipid loading following CD36 silencing mediated by thrombin-activated platelet delivery. Scale bar = 20 μm. n = 3. (M − N) Western blot analysis and quantification showing increased Cyclin E1 expression following platelet-mediated siCD36 delivery. n = 3. (O-P) Western blot analysis and quantification showing reduced global H3K4me3 levels following CD36 silencing, consistent with altered chromatin state associated with metabolic reprogramming. n = 3. (Q-V) Immunostaining and quantification of cell cycle-associated markers Ki67, phosphorylated histone H3 (pH3), and Aurora B kinase following thrombin-activated delivery. Scale bar = 20 μm. n = 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns = non-significant.

Article Snippet: Three siRNA duplexes targeting different regions of the murine CD36 gene were synthesized (Synthace),And the sequences were provided in the supply.

Techniques: Encapsulation, Activity Assay, Transmission Assay, Electron Microscopy, Disruption, Confocal Microscopy, Labeling, In Vitro, Imaging, Western Blot, Knockdown, Immunofluorescence, Expressing, Staining, Immunostaining

Journal: iScience

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1016/j.isci.2026.115821

Figure Lengend Snippet: TBEV NS5 interacts with P300 to modulate G0/G1 cell cycle progression (A) Cells were transfected with the plasmids of Flag-tagged EV and ten TBEV viral proteins, the cell distribution was analyzed by flow cytometry. (B) The expression plasmids of Flag-EV, 0.5 and 1.0 μg Flag-NS5 were transfected into A549 cells, the cell distribution was analyzed by flow cytometry. The experiment was repeated for three times, and the percentages of cells were shown in column graph. (C) Cells were transfected with Flag-EV or Flag NS5 together with HA-P300, the interaction between NS5 and P300 was analyzed by co-immunoprecipitation analysis. (D) The colocalization of P300 (green) and NS5 (red) were examined by immunofluorescence analysis. Scale bars, 10 μm. (E–G) Cells transfected with NS5 were collected after 48 h, the expression of CDK4, CDK6, and P16 was analyzed by immunoblot (E). (F) Quantification of protein levels from (E). Data are normalized to actin and presented as fold change relative to control (mean ± SD, n = 3). (G) qPCR analysis of mRNA expression of CDK4, CDK6, and P16 upon NS5 overexpression. Data are normalized to GAPDH using the 2ˆ(-ΔΔCt) method and shown as fold change relative to control (mean ± SD, n = 3). (H) A549 cells transfected with HA-EV, HA P300 and P300 Hm were further transfected with NS5, the distribution of cells was analyzed by flow cytometry. The experiments were repeated for three times, and the proportions of cells were shown in column graph, the expression of indicated proteins was assessed by immunoblot assay. (I) Cells transfected with P300 siRNA and NC siRNA (siNC) were further transfected with NS5, the distribution of cells was analyzed by flow cytometry, and the proportions of cells and the relative expression of P300 mRNA were shown in column graph. (J) Cells transfected with HA-EV, HA P300, and P300 Hm were mock-infected or infected TBEV, the distribution of cells was analyzed by flow cytometry. (K) Cells transfected with P300 siRNA and NC siRNA were mock-infected or infected TBEV, the distribution of cells was analyzed by flow cytometry. (L) Outline of G0/G1 cell-cycle arrest regulated by TBEV NS5. Data in column graphs are represented as mean ± SEM of three independent experiments. The p values are calculated and reported using one-way ANOVA.

Article Snippet: For small interfering RNA (siRNA) targeting P300, 5′-GGACUACCCUAUCAAGUAATT-3′, was synthesized at Sangon Biotech (Shanghai, China).

Techniques: Transfection, Flow Cytometry, Expressing, Immunoprecipitation, Immunofluorescence, Western Blot, Control, Over Expression, Infection

Journal: iScience

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1016/j.isci.2026.115821

Figure Lengend Snippet: P300 inhibitors and CDK4 agonist restrict viral gene expression by interfering with TBEV-induced cell-cycle arrest (A and B) A549 cells were either mock-infected or infected with TBEV, the cells were then treated with indicated concentration of C646 (A) and CPI-637 (B), the supernatants were collected at 48 hpi, and the mRNA level of TBEV E gene was analyzed by probe qPCR. (C–F) Cells mock-infected or infected with TBEV at the MOI of ten were treated with 10 μM C646 and CPI-637, the expression of TBEV NS1 protein was detected by immunoblot (C and D) and immunofluorescence analysis (E), and the cell cycle distribution was analyzed by flow cytometry (F). Scale bars, 50 μm. (G) Cells transfected with HA-EV, HA P300, and P300Hm were infected TBEV, the mRNA level of TBEV E gene in the supernatants was analyzed by probe qPCR. (H) Cells transfected with P300 siRNA and NC siRNA were infected TBEV, the mRNA level of TBEV E gene in the supernatants was analyzed by probe qPCR. (I) A549 cells were treated with the indicated concentrations of chrysin for 2 h, the cells were then infected with TBEV, the supernatants were collected at 48 hpi, and the mRNA level of TBEV E gene was analyzed by probe qPCR. (J–L) Cells were treated with 10 μM chrysin for 2 h, the cells were then mock-infected or infected with TBEV at the MOI of ten, the expression of TBEV NS1 protein was detected by immunoblot (J), and the cell distribution was analyzed by flow cytometry (K). The percentage of cells (L) upon chrysin treatment was shown in column graphs. Scale bars, 50 μm. Data in column graphs are represented as mean ± SEM of three independent experiments. The p values are calculated and reported using one-way ANOVA.

Article Snippet: For small interfering RNA (siRNA) targeting P300, 5′-GGACUACCCUAUCAAGUAATT-3′, was synthesized at Sangon Biotech (Shanghai, China).

Techniques: Gene Expression, Infection, Concentration Assay, Expressing, Western Blot, Immunofluorescence, Flow Cytometry, Transfection