nih3t3  (ATCC)

Journal: Aging and Disease

Article Title: The Flip Side of the Coin: METTL3 Serves as a Novel Cellular Senescence Accelerator via Negative Regulation of ITGA9

doi: 10.14336/AD.2024.1715

Figure Lengend Snippet: M6A modifications within total mRNA were significantly enhanced during the aging process . ( A ) Left: representative photographs of 2-month and 20-month-old male C57BL/6 mouse; Right: representative photographs of SA-β-gal staining in kidney tissues from young and old C57-BL/6 mice. ( B ) Left: representative photographs of SA-β-gal staining of P3 and P9 MEF cells; Right: the p16 expression levels in P3 and P9 MEF cells. ( C ) Left: representative photographs of SA-β-gal staining of H 2 O 2 -induced premature senescence of NIH/3T3 cells; Right: the p16 expression levels in H 2 O 2 treated NIH/3T3 cells. ( D ) Poly(A)+ RNA was extracted from the brain and liver tissue of young and old C57-BL/6 mice and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( E ) Poly(A)+ RNA was extracted from the P3 and P9 MEF cells and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( F ) Poly(A)+ RNA was extracted from the P3 and P9 MEF cells and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( G ) Expression levels of m6A modification associated proteins were measured in P3 and P9 MEF cells, and the quantitative results were shown on the right. Mean (±SEM), n=4, passed normality test: Shapiro-Wilk test; One sample t test (MEF P9 vs 1, METTL3: p =0.0027, t = 9.176).

Article Snippet: NIH/3T3 fibroblasts were procured from the American Type Culture Collection (ATCC).

Techniques: Staining, Expressing, Dot Blot, Control, Modification

Journal: Animal Cells and Systems

Article Title: Quercus infectoria gall extract modulates adipocyte differentiation and lipid metabolism through dual regulation of adipogenesis and lipolysis in 3T3-L1 adipocytes

doi: 10.1080/19768354.2026.2623321

Figure Lengend Snippet: Time-dependent inhibition of adipogenesis and lipid accumulation by Quercus infectoria gall (QIG) extract in 3T3-L1 cells. D/A and M/M are abbreviated for differentiation/activation medium and maintenance medium, respectively. (a) Schematic representation of the experimental timeline. (b) Representative images of Oil Red O staining for lipid accumulation in 3T3-L1 cells at the end of the differentiation (day 8). Images include preadipocytes, adipocytes, and cells treated with QIG extract at various time points during adipogenesis, as indicated in (a). Scale bar = 100 µm. (c) Relative lipid accumulation in 3T3-L1 adipocytes, as determined by Oil Red O staining shown in (b). Data represent three independent experiments performed in triplicate and are expressed as mean ± SD (error bars). Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: Cell culture, maintenance, and differentiation of 3T3-L1 preadipocytes (ATCC® CL-173TM, Manassas, VA, USA; CVCL_0123) were conducted as previously described (Jakkawanpitak et al. ).

Techniques: Inhibition, Activation Assay, Staining

Journal: Animal Cells and Systems

Article Title: Quercus infectoria gall extract modulates adipocyte differentiation and lipid metabolism through dual regulation of adipogenesis and lipolysis in 3T3-L1 adipocytes

doi: 10.1080/19768354.2026.2623321

Figure Lengend Snippet: Quercus infectoria gall (QIG) extract promotes lipolysis and modulates lipolytic protein expression in 3T3-L1 adipocytes. (a) Experimental timeline illustrating the stages of adipocyte differentiation and QIG treatment. (b) Cell viability of 3T3-L1 adipocytes after 48 h of treatment with QIG extract at various concentrations. (c) Relative lipolytic activity of adipocytes treated with QIG extract (6.25–100 µg/mL), as determined by glycerol release assay. (d) Representative Western blot data showing the expression of p-HSL (S660), total HSL, ATGL, and β-actin in adipocytes after 48 h of treatment. (e) Quantification of protein expression shown in (d). Data represent three independent experiments performed in triplicate and are expressed as mean ± SD (error bars). Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: Cell culture, maintenance, and differentiation of 3T3-L1 preadipocytes (ATCC® CL-173TM, Manassas, VA, USA; CVCL_0123) were conducted as previously described (Jakkawanpitak et al. ).

Techniques: Expressing, Activity Assay, Glucose Assay, Western Blot

Journal: Animal Cells and Systems

Article Title: Quercus infectoria gall extract modulates adipocyte differentiation and lipid metabolism through dual regulation of adipogenesis and lipolysis in 3T3-L1 adipocytes

doi: 10.1080/19768354.2026.2623321

Figure Lengend Snippet: Quercus infectoria gall (QIG) extract inhibits adipogenesis and reduces lipid accumulation in a dose-dependent manner. (a) Cell viability of 3T3-L1 preadipocytes treated with increasing concentrations of QIG extract (3.125–200 µg/mL) for 24 h, assessed using the MTT assay. (b) Representative images of Oil Red O staining for lipid accumulation in 3T3-L1 cells. Scale bar = 100 µm. (c) Relative lipid accumulation in cells treated with QIG extract (3.125–25 µg/mL) during differentiation, as determined by Oil Red O staining. Data represent three independent experiments performed in triplicate and are expressed as mean ± SD (error bars). Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: Cell culture, maintenance, and differentiation of 3T3-L1 preadipocytes (ATCC® CL-173TM, Manassas, VA, USA; CVCL_0123) were conducted as previously described (Jakkawanpitak et al. ).

Techniques: MTT Assay, Staining

Journal: Animal Cells and Systems

Article Title: Quercus infectoria gall extract modulates adipocyte differentiation and lipid metabolism through dual regulation of adipogenesis and lipolysis in 3T3-L1 adipocytes

doi: 10.1080/19768354.2026.2623321

Figure Lengend Snippet: Inhibitory effects of Quercus infectoria gall (QIG) extract and its phenolic constituents on adipocyte differentiation in 3T3-L1 preadipocytes. (a) Dose-dependent inhibition of lipid accumulation by QIG extract and its phenolic components (tannic acid, ellagic acid, and gallic acid) in 3T3-L1 cells. Orlistat was included as a positive control for an anti-obesity drug. Relative lipid accumulation in cells was determined by Oil Red O staining and expressed as a percentage of the adipocyte control. (b) Representative Oil Red O staining images of preadipocytes, adipocytes, and cells treated during adipogenesis with orlistat, QIG extract, tannic acid, ellagic acid, or gallic acid (25 µg/mL each). Scale bar = 100 µm. (c) Relative lipid accumulation in cells, as determined by Oil Red O staining shown in (b). Data represent three independent experiments performed in triplicate and are expressed as mean ± SD (error bars). Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: Cell culture, maintenance, and differentiation of 3T3-L1 preadipocytes (ATCC® CL-173TM, Manassas, VA, USA; CVCL_0123) were conducted as previously described (Jakkawanpitak et al. ).

Techniques: Inhibition, Positive Control, Staining, Control

Journal: Animal Cells and Systems

Article Title: Quercus infectoria gall extract modulates adipocyte differentiation and lipid metabolism through dual regulation of adipogenesis and lipolysis in 3T3-L1 adipocytes

doi: 10.1080/19768354.2026.2623321

Figure Lengend Snippet: Quercus infectoria gall (QIG) extract suppresses mitotic clonal expansion during early adipogenic differentiation of 3T3-L1 preadipocytes. Differentiation was initiated by adding differentiation/activation medium (D/A) to 3T3-L1 preadipocytes in the presence or absence of QIG extract at concentrations of 3.12, 6.25, 12.5, and 25 μg/mL. (a) Representative flow cytometry histograms showing the cell cycle distribution after 18 h of treatment. (b) Percentage of the cell population in the G0/G1, S, and G2/M phases, as quantified from the data in (a). (c) Cell proliferation analysis of preadipocytes, differentiating adipocytes, and QIG-treated differentiating adipocytes over 48 h. (d) mRNA expression levels of DNA replication initiation and mitotic clonal expansion–associated genes ( Cdc45 , Mcm3 , and Gins1 ) in differentiating 3T3-L1 adipocytes treated with the indicated concentrations of QIG extract. Data represent three independent experiments performed in triplicate and are expressed as mean ± SD (error bars). Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: Cell culture, maintenance, and differentiation of 3T3-L1 preadipocytes (ATCC® CL-173TM, Manassas, VA, USA; CVCL_0123) were conducted as previously described (Jakkawanpitak et al. ).

Techniques: Activation Assay, Flow Cytometry, Expressing

Journal: Animal Cells and Systems

Article Title: Quercus infectoria gall extract modulates adipocyte differentiation and lipid metabolism through dual regulation of adipogenesis and lipolysis in 3T3-L1 adipocytes

doi: 10.1080/19768354.2026.2623321

Figure Lengend Snippet: Quercus infectoria gall (QIG) extract suppresses adipogenic and lipogenic gene expression in differentiating 3T3-L1 adipocytes. Differentiation was initiated by culturing 3T3-L1 preadipocytes in differentiation/activation (D/A) medium with or without QIG extract (3.12–25 μg/mL). Gene expression levels were determined by RT-qPCR after 48 h of adipogenic induction. (a–d) mRNA expression of adipogenic transcription factors and adipocyte marker genes: Cebpa (a), Pparg (b), Fabp4 (c), and Adipoq (d). (e–g) mRNA expression of lipogenesis-related genes: Fas (e), Srebf1 (f), and Lpl (g). (h) Heatmap depicting the relative mRNA expression levels corresponding to the data presented in (a–g). Data represent three independent experiments performed in triplicate and are expressed as mean ± SD (error bars). Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: Cell culture, maintenance, and differentiation of 3T3-L1 preadipocytes (ATCC® CL-173TM, Manassas, VA, USA; CVCL_0123) were conducted as previously described (Jakkawanpitak et al. ).

Techniques: Gene Expression, Activation Assay, Quantitative RT-PCR, Expressing, Marker

Journal: Animal Cells and Systems

Article Title: Quercus infectoria gall extract modulates adipocyte differentiation and lipid metabolism through dual regulation of adipogenesis and lipolysis in 3T3-L1 adipocytes

doi: 10.1080/19768354.2026.2623321

Figure Lengend Snippet: Quercus infectoria gall (QIG) extract inhibits the expression of key adipogenic transcription factors and enzymes in differentiating 3T3-L1 adipocytes in a dose-dependent manner. The differentiation/activation medium (D/A) was added to the 3T3-L1 preadipocytes with or without QIG extract at varying concentrations (3.12, 6.25, 12.5, and 25 μg/mL). The treatment was performed for 3 days before harvesting the cells for Western blot assays. (a) Representative Western blot data set performed using anti-C/EBPβ, anti-C/EBPα, anti-PPARγ, anti-FAS, and anti-β-actin antibodies. (b) Quantification of the levels of C/EBPβ, C/EBPα, PPARγ, and FAS proteins, as shown in (a). Data represent three independent experiments performed in triplicate and are expressed as mean ± SD (error bars). Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: Cell culture, maintenance, and differentiation of 3T3-L1 preadipocytes (ATCC® CL-173TM, Manassas, VA, USA; CVCL_0123) were conducted as previously described (Jakkawanpitak et al. ).

Techniques: Expressing, Activation Assay, Western Blot

Journal: Animal Cells and Systems

Article Title: Quercus infectoria gall extract modulates adipocyte differentiation and lipid metabolism through dual regulation of adipogenesis and lipolysis in 3T3-L1 adipocytes

doi: 10.1080/19768354.2026.2623321

Figure Lengend Snippet: Quercus infectoria gall (QIG) extract regulates lipid accumulation and reduces lipid droplet size in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes (8 days post-differentiation) were treated with or without QIG extract at concentrations of 12.5, 25, 50, and 100 μg/mL. The medium was refreshed every 2 days until day 6, after which the cells were stained with Oil Red O. (a) Cell viability of 3T3-L1 adipocytes following 6 days of treatment with QIG extract at the indicated concentrations. (b) Representative images showing lipid accumulation in preadipocytes, mature adipocytes (control), and QIG-treated adipocytes at increasing concentrations (12.5–100 μg/mL). Scale bar = 100 µm. (c) Quantification of lipid accumulation corresponding to the images shown in (b). Data represent three independent experiments performed in triplicate and are expressed as mean ± SD (error bars). Different letters indicate statistically significant differences ( p < 0.05). (d) Comparison of lipid droplet size in mature adipocytes and those treated with 100 µg/mL QIG extract. Scale bar = 50 µm. A histogram of lipid droplet size distribution was generated from measurements of 150 cells per group, obtained from three independent microscopic images.

Article Snippet: Cell culture, maintenance, and differentiation of 3T3-L1 preadipocytes (ATCC® CL-173TM, Manassas, VA, USA; CVCL_0123) were conducted as previously described (Jakkawanpitak et al. ).

Techniques: Staining, Control, Comparison, Generated

Journal: Genes & Development

Article Title: Plagl1 and Lrrc58 control mammalian body size by triggering target-directed microRNA degradation of miR-322 and miR-503

doi: 10.1101/gad.353138.125

Figure Lengend Snippet: Validation of novel TDMD triggers. ( A ) Schematic of trigger site deletion (Δts) strategy to validate putative TDMD triggers. ( B , D ) Genomic organization of TDMD trigger transcripts with conservation and predicted miRNA base-pairing architecture of the trigger sites. Nucleotides predicted to base-pair with the miRNA are shown in bold. ( C , E ) qRT-PCR analysis of indicated miRNAs, relative to miR-16-5p, in WT and Δts MEFs. Parental MEFs or two independent Δts clones for each trigger site were infected with lentivirus expressing Cas9 and a nontargeting CRISPR guide (sgNT) or Zswim8- targeting guide (sg Zswim8 ). Values were normalized to expression level in sg Zswim8 for each condition. n = 3 technical replicates per clone, with individual data points plotted (mean ± SD shown). P -values were calculated by one-tailed Student's t -test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. ( F ) qRT-PCR analysis of the indicated miRNAs, relative to U6, in NIH3T3 cells at time points after serum starvation and restimulation. NIH3T3 cells expressing dCas9-KRAB were infected with lentivirus encoding a nontargeting guide (sgNT) or guides targeting Plagl1 or Lrrc58 . Parental NIH3T3 cells were infected with lentivirus expressing Cas9 and a nontargeting guide (sgNT) or Zswim8- targeting guide (sg Zswim8 ). Values were normalized to expression at the 0 h time point for each condition. n = 3 technical replicates per condition, with mean ± SD shown. P -values were calculated by one-tailed Student's t -test comparing sg Plagl1 (dCas9-KRAB) or sg Lrrc58 (dCas9-KRAB) with sgNT (dCas9-KRAB) or comparing sg Zswim8 (Cas9) with sgNT (Cas9). (*) P < 0.05.

Article Snippet: HEK293T and NIH3T3 cells were obtained from ATCC, and immortalized MEFs were generated previously by transfecting primary MEFs with a plasmid expressing SV40 large T antigen ( ).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Clone Assay, Infection, Expressing, CRISPR, One-tailed Test