Journal: Nature Communications

Article Title: Circular RNA-mediated inverse prime editing in human cells

doi: 10.1038/s41467-025-59120-7

Figure Lengend Snippet: a Schematic diagram of circular RNA-mediated inverse prime editors (ciPEs). b Schematic diagrams of the structure of ciPE editors. CMV, the CMV promoter of cytomegalovirus; U6, the polymerase III promoter of U6; NLS, bipartite nuclear localization signal; M-MLV RTΔRNase H, deletion variant of M-MLV RT with no RNase H domain; 5′ RL, 5′ ribozyme and ligation sequences; 3′ RL, 3′ ribozyme and ligation sequences; RTT, reverse transcriptase template; PBS, primer binding site. c Comparison of inverse prime editing frequencies between split iPE2 and ciPE2 at four target sites in HEK293T cells. Frequencies (mean ± s.e.m.) in c were obtained from three biological replicates ( n = 3). d Comparison of inverse prime editing efficiencies between ciPE2–5 and split iPEmax2–5 using epegRNA at six target sites in HEK293T cells. Frequencies (mean ± s.e.m.) in d were obtained from four biological replicates ( n = 4). circRNA, circular RNA; Ins, insertion; Del, deletion. InDels, byproducts of random insertions and deletions. P values were obtained from two-tailed Student’s t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: The sgRNA, nicking sgRNA, pegRNA, and epegRNA vectors driven by the human U6 promoter were constructed by annealing oligonucleotides and inserting them into the pOsU3 backbone (Addgene #170132) using the Golden Gate assembly method.

Techniques: Variant Assay, Ligation, Reverse Transcription, Binding Assay, Comparison, Two Tailed Test

Journal: Discover Oncology

Article Title: Integrated prognostic assessment of apoptosis and chemotherapy related gene in bladder cancer: a prognostic signature

doi: 10.1007/s12672-025-02581-5

Figure Lengend Snippet: Validation of Gemcitabine resistance in BC cell lines. A CCK-8 assay was performed to detect cell viability treated with different concentrations of gemcitabine in T24, UMUC3, T24-Gem and UMUC3-Gem cells. B The protein levels of FASN, GLI2, PDGFRA and VHL in T24, T24-Gem, UMUC3, and UMUC3-Gem cells were validated by western blotting. C , D The efficiency of FASN silencing in T24-Gem and UMUC3-Gem cells was detected by qRT-PCR and western blotting. E Cell viability abilities of T24-Gem and UMUC3-Gem cells treated with different concentrations of Gemcitabine at 72 h after transfection were evaluated by CCK-8 assays. Data are presented as mean ± Standard Error of Mean (SEM) from three independent replicates. * P < 0.05, ** P < 0.01 (Student’s t test)

Article Snippet: After a 1-h blocking step at room temperature, primary antibodies targeting human FASN, GLI2, PDGFRA, VHL or β-Actin from Proteintech Group, Wuhan, China, were incubated at 4 °C for 12 h. FASN (Cat. No. 10624-2-AP), GLI2 (Cat. No. 18989-1-AP) and β-Actin (Cat. No. 81115-1-RR) were purchased from Proteintech Group (Wuhan, China).

Techniques: Biomarker Discovery, CCK-8 Assay, Western Blot, Quantitative RT-PCR, Transfection

Journal: Discover Oncology

Article Title: Integrated prognostic assessment of apoptosis and chemotherapy related gene in bladder cancer: a prognostic signature

doi: 10.1007/s12672-025-02581-5

Figure Lengend Snippet: Validation of Cisplatin resistance in BC cell lines. A CCK-8 assay was performed to detect cell viability treated with different concentrations of Cisplatin in T24, UMUC3, T24-CDDP and UMUC3-CDDP cells. B The protein levels of FASN, GLI2, PDGFRA and VHL in T24, T24-CDDP, UMUC3, and UMUC3-CDDP cells were validated by western blotting. C , D Cell viability abilities of T24-Gem and UMUC3-Gem cells treated with different concentrations of Cisplatin at 72 h after transfection were evaluated by CCK-8 assays. Data are presented as mean ± Standard Error of Mean (SEM) from three independent replicates

Article Snippet: After a 1-h blocking step at room temperature, primary antibodies targeting human FASN, GLI2, PDGFRA, VHL or β-Actin from Proteintech Group, Wuhan, China, were incubated at 4 °C for 12 h. FASN (Cat. No. 10624-2-AP), GLI2 (Cat. No. 18989-1-AP) and β-Actin (Cat. No. 81115-1-RR) were purchased from Proteintech Group (Wuhan, China).

Techniques: Biomarker Discovery, CCK-8 Assay, Western Blot, Transfection

Journal: Life Science Alliance

Article Title: Fatty acid synthase inhibition alleviates lung fibrosis via β-catenin signal in fibroblasts

doi: 10.26508/lsa.202402805

Figure Lengend Snippet: (A) Representative images and statistical analysis of double-labeled immunofluorescence staining for FASN and α-SMA in lung sections from IPF patients (n = 3) and donors (n = 3). (B) Representative images and statistical analysis of double-labeled immunofluorescence staining for FASN and α-SMA in lung sections from bleomycin-induced fibrotic mouse (n = 6) and saline controls (n = 6). Figures inside the white box were zoomed in on the rightmost. (C, D) Western blotting analysis of FASN protein expression in MRC-5 cells stimulated with or without TGF-β1 (10 ng/ml) for 24 h. n = 3. (E) Quantitative RT–PCR analysis of relative FASN mRNA expression of MRC-5 cells stimulated with or without TGF-β1 (10 ng/ml) for 12 h. n = 3. (F, G) Western blotting analysis of FASN protein expression in primary lung fibroblasts isolated from bleomycin-induced fibrotic mouse and saline mouse. n = 3. (H) Quantitative RT–PCR analysis of the relative FASN mRNA expression of primary lung fibroblasts isolated from bleomycin-induced fibrotic mouse and saline mouse. n = 3. Source data are available for this figure.

Article Snippet: Human FASN shRNA (Targeted 5′-CCTACTGGATGCGTTCTTCAA-3′) and mouse FASN shRNA (Targeted 5′-GCTGGTCGTTTCTCCATTAAA-3′) were designed and synthesized by Sangon Biotech and subsequently annealed and inserted into the pLKO.1.

Techniques: Labeling, Immunofluorescence, Staining, Saline, Western Blot, Expressing, Quantitative RT-PCR, Isolation

Journal: Life Science Alliance

Article Title: Fatty acid synthase inhibition alleviates lung fibrosis via β-catenin signal in fibroblasts

doi: 10.26508/lsa.202402805

Figure Lengend Snippet: (A) Images show the migration area of MRC-5 cells by the wound-healing assay after FASN shRNA (n = 3) or NC (n = 3). (B) Images show transmigration cells down to the Transwell chamber with FASN shRNA or NC. (C) Representative image of immunofluorescence staining for PLIN2 in MRC-5 cells by FASN shRNA (n = 3) or NC (n = 3). (D) Representative image of immunofluorescence staining for collagen 1 in MRC-5 cells by FASN shRNA (n = 3) or NC (n = 3). (E, F) Western blotting analysis of the protein expression of FASN, fibronectin, collagen 1, α-SMA, and PLIN2 in MRC-5 cells by FASN shRNA (n = 3) or NC (n = 3). (G) Quantitative RT–PCR analysis of relative FASN , fibronectin , collagen 1 , ACTA2 , and PLIN2 mRNA expression in MRC-5 cells with FASN shRNA (n = 3) or NC (n = 3). (H) Images show the migration area of MRC-5 cells by the wound-healing assay by C75 (30 μM, n = 3) for 36 h or not (n = 3). (I) Images show transmigration cells down to the Transwell chamber with C75 (30 μM, n = 3) for 48 h or not (n = 3). (J) Representative images of immunofluorescence staining for PLIN2 in MRC-5 cells by C75 (30 μM, n = 3) for 48 h or not (n = 3). (K) Representative images of immunofluorescence staining for collagen 1 in MRC-5 cells by C75 (30 μM, n = 3) for 48 h or not (n = 3). (L, M) Western blotting analysis of the protein expression of fibronectin, collagen 1, α-SMA, and PLIN2 in MRC-5 cells by C75 (30 μM, n = 3) for 48 h or not (n = 3). (N) Quantitative RT–PCR analysis of relative fibronectin , collagen 1 , ACTA2 , and PLIN2 mRNA expression in MRC-5 cells without or with C75 (30 μM, n = 3) for 12 h or not (n = 3). Source data are available for this figure.

Article Snippet: Human FASN shRNA (Targeted 5′-CCTACTGGATGCGTTCTTCAA-3′) and mouse FASN shRNA (Targeted 5′-GCTGGTCGTTTCTCCATTAAA-3′) were designed and synthesized by Sangon Biotech and subsequently annealed and inserted into the pLKO.1.

Techniques: Migration, Wound Healing Assay, shRNA, Transmigration Assay, Immunofluorescence, Staining, Western Blot, Expressing, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: Fatty acid synthase inhibition alleviates lung fibrosis via β-catenin signal in fibroblasts

doi: 10.26508/lsa.202402805

Figure Lengend Snippet: (A) Representative images of immunofluorescence staining illustrating the co-localization of overexpression (OE)-β-catenin (n = 3) or not OE-β-catenin (n = 3) and FASN in MRC-5 cells. (B, C) Western blotting analysis of FLAG and FASN expression in MRC-5 cells by OE-β-catenin by Phage-FLAG-β-catenin or not. (D) Quantitative RT–PCR analysis of relative β-catenin and FASN mRNA expression in MRC-5 cells by OE-β-catenin (n = 3) or not (n = 3). (E, F) Western blotting analysis of β-catenin and FASN expression in HEK293T cells by OE-β-catenin (n = 3) or not (n = 3). (G) Quantitative RT–PCR analysis of relative β-catenin and FASN mRNA expression in HEK293T cells by OE-β-catenin (n = 3) or not (n = 3). (H) Immunoprecipitation showed that FASN interacted with β-catenin, GSK3B, and Axin1 in HEK293T cells, but not with CK1a. Source data are available for this figure.

Article Snippet: Human FASN shRNA (Targeted 5′-CCTACTGGATGCGTTCTTCAA-3′) and mouse FASN shRNA (Targeted 5′-GCTGGTCGTTTCTCCATTAAA-3′) were designed and synthesized by Sangon Biotech and subsequently annealed and inserted into the pLKO.1.

Techniques: Immunofluorescence, Staining, Over Expression, Western Blot, Expressing, Quantitative RT-PCR, Immunoprecipitation

Journal: Life Science Alliance

Article Title: Fatty acid synthase inhibition alleviates lung fibrosis via β-catenin signal in fibroblasts

doi: 10.26508/lsa.202402805

Figure Lengend Snippet: (A) Representative images of immunofluorescence staining for β-catenin in MRC-5 cells by FASN shRNA or NC and treated with Ladu (5 ng/ml) for 24 h. n = 3. (B, C) Western blotting analysis of the protein expression of FASN and β-catenin in MRC-5 cells by FASN shRNA (n = 3) or NC (n = 3). (D, E, F) Western blotting analysis of β-catenin expression in the cytoplasm and in the nucleus in MRC-5 cells by FASN shRNA (n = 3) or NC (n = 3). (G) Representative images of immunofluorescence staining for β-catenin in MRC-5 cells by C75 (30 μM) for 24 h and then treated with Ladu (5 ng/ml) for 24 h. n = 3. (H, I) Western blotting analysis of the protein expression of β-catenin in MRC-5 cells by C75 (30 μM, n = 3) for 48 h or not (n = 3). (J, K, L) Western blotting analysis of β-catenin expression in the cytoplasm and in the nucleus in MRC-5 cells with or without C75 (30 μM) for 48 h. n = 3. (M) Quantitative RT–PCR analysis of the relative mRNA expression of FASN and β-catenin in MRC-5 cells by FASN shRNA (n = 3) or NC (n = 3). (N) Quantitative RT–PCR analysis of the relative mRNA expression of β-catenin in MRC-5 cells by C75 (30 μM, n = 3) for 12 h or not (n = 3). Source data are available for this figure.

Article Snippet: Human FASN shRNA (Targeted 5′-CCTACTGGATGCGTTCTTCAA-3′) and mouse FASN shRNA (Targeted 5′-GCTGGTCGTTTCTCCATTAAA-3′) were designed and synthesized by Sangon Biotech and subsequently annealed and inserted into the pLKO.1.

Techniques: Immunofluorescence, Staining, shRNA, Western Blot, Expressing, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: Fatty acid synthase inhibition alleviates lung fibrosis via β-catenin signal in fibroblasts

doi: 10.26508/lsa.202402805

Figure Lengend Snippet: (A, B) Western blotting analysis of the protein expression of FASN and β-catenin in mouse lung fibroblasts by FASN shRNA (n = 3) or NC (n = 3). (C) Quantitative RT–PCR analysis of the relative mRNA expression of FASN and β-catenin in mouse lung fibroblasts by FASN shRNA (n = 3) or NC (n = 3). (D, E) Western blotting analysis of the protein expression of β-catenin in mouse lung fibroblasts by C75 (30 μM, n = 3) for 48 h or not (n = 3). (F) Quantitative RT–PCR analysis of the relative mRNA expression of β-catenin in mouse lung fibroblasts by C75 (30 μM, n = 3) for 12 h or not (n = 3). Source data are available for this figure.

Article Snippet: Human FASN shRNA (Targeted 5′-CCTACTGGATGCGTTCTTCAA-3′) and mouse FASN shRNA (Targeted 5′-GCTGGTCGTTTCTCCATTAAA-3′) were designed and synthesized by Sangon Biotech and subsequently annealed and inserted into the pLKO.1.

Techniques: Western Blot, Expressing, shRNA, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: Fatty acid synthase inhibition alleviates lung fibrosis via β-catenin signal in fibroblasts

doi: 10.26508/lsa.202402805

Figure Lengend Snippet: (A) Quantitative RT–PCR analysis of the relative mRNA expression levels of FASN , GSK3B , and Axin1 in MRC-5 cells transduced with FASN shRNA (n = 3) or NC (n = 3). (B) Quantitative RT–PCR analysis of the relative mRNA expression levels of GSK3B and Axin1 in MRC-5 cells after treatment with C75 (30 μM) for 12 h. n = 3. (C) Western blot analysis of GSK3B and FASN protein expression in MRC-5 cells from the FASN shRNA (n = 3) or NC groups (n = 3). (D) Western blot analysis of the relative protein expression level of GSK3B in MRC-5 cells after treatment with C75 (30 μM) for 24 h. n = 3. (E) Western blot analysis of Axin1 and FASN protein expression in MRC-5 cells from the FASN shRNA (n = 3) or NC groups (n = 3). (F) Western blot analysis of the relative protein expression level of Axin1 in MRC-5 cells after treatment with C75 (30 μM) for 24 h. n = 3. (G) Quantitative RT–PCR analysis of the relative mRNA expression levels of FASN , GSK3B , and Axin1 in HEK293T cells transduced with FASN shRNA (n = 3) or NC (n = 3). (H) Quantitative RT–PCR analysis of the relative mRNA expression levels of GSK3B and Axin1 in HEK293T cells after treatment with C75 (30 μM) for 12 h. n = 3. (I) Western blot analysis of GSK3B and FASN protein expression in HEK293T cells from the FASN shRNA (n = 3) or NC groups (n = 3). (J) Western blot analysis of the relative protein expression level of GSK3B in HEK293T cells after treatment with C75 (30 μM) for 24 h. n = 3. (K) Western blot analysis of Axin1 and FASN protein expression in HEK293T cells from the FASN shRNA (n = 3) or NC groups (n = 3). (L) Western blot analysis of the relative protein expression level of Axin1 in HEK293T cells after treatment with C75 (30 μM) for 24 h. n = 3. GSK3B or Axin1 was overexpressed using phage-FLAG-GSK3B or phage-FLAG-Axin1. Flag-tagged proteins indicate the corresponding protein levels. Source data are available for this figure.

Article Snippet: Human FASN shRNA (Targeted 5′-CCTACTGGATGCGTTCTTCAA-3′) and mouse FASN shRNA (Targeted 5′-GCTGGTCGTTTCTCCATTAAA-3′) were designed and synthesized by Sangon Biotech and subsequently annealed and inserted into the pLKO.1.

Techniques: Quantitative RT-PCR, Expressing, Transduction, shRNA, Western Blot

Journal: Life Science Alliance

Article Title: Fatty acid synthase inhibition alleviates lung fibrosis via β-catenin signal in fibroblasts

doi: 10.26508/lsa.202402805

Figure Lengend Snippet: (A, B) Representative images of the EdU assay and analysis of MRC-5 cells with FASN shRNA (n = 3) or NC (n = 3). (C) Bar graph showed the cell viability measured by CCK8 with FASN shRNA (n = 3) or NC (n = 3). (D) Western blotting analysis of β-catenin protein degradation after treatment with 100 μg/ml CHX for different lengths of time in MRC-5 cells than pre-treatment with FASN shRNA (n = 3) or NC (n = 3). (E) Western blotting analysis of the protein expression of LC3 II/LC3 I in MRC-5 cells treated with CQ (50 μM) for 4 h after pre-treatment with FASN shRNA (n = 3) or NC (n = 3). (F) Immunofluorescence staining of GFP/RFP-LC3 in MRC-5 cells treated with CQ (50 μM) for 4 h after pre-treatment with FASN shRNA (n = 3) or NC (n = 3). (G, H) Representative images of the EdU assay and analysis of MRC-5 cells with 30 μM C75 (n = 3) for 24 h or not (n = 3). (I) Bar graph showed the cell viability measured by CCK8 with C75 (n = 3) for 24 h or not (n = 3). (J) Western blotting analysis of β-catenin protein degradation after treatment with 100 μg/ml CHX for different lengths of time in MRC-5 cells than pre-treatment with C75 (n = 3) for 24 h or not (n = 3). (K) Western blotting analysis of the protein expression of LC3 II/LC3 I in MRC-5 cells treated with CQ (50 μM) for 4 h after pre-treatment with C75 (n = 3) for 24 h or not (n = 3). (L) Immunofluorescence staining of GFP/RFP-LC3 in MRC-5 cells treated with (CQ, 50 μM) for 4 h after pre-treatment with C75 (n = 3) for 24 h or not (n = 3). Source data are available for this figure.

Article Snippet: Human FASN shRNA (Targeted 5′-CCTACTGGATGCGTTCTTCAA-3′) and mouse FASN shRNA (Targeted 5′-GCTGGTCGTTTCTCCATTAAA-3′) were designed and synthesized by Sangon Biotech and subsequently annealed and inserted into the pLKO.1.

Techniques: EdU Assay, shRNA, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Life Science Alliance

Article Title: Fatty acid synthase inhibition alleviates lung fibrosis via β-catenin signal in fibroblasts

doi: 10.26508/lsa.202402805

Figure Lengend Snippet: (A) Statistical analysis of bar charts showed the protein expression level of LC3 II/LC3 I in MRC-5 cells treated with CQ (50 μM) for 4 h after pre-treatment with FASN shRNA or NC. n = 3. (B) Statistical analysis of bar charts showed the protein expression level of LC3 II/LC3 I in MRC-5 cells treated with CQ (50 μM) for 4 h after pre-treatment with C75 or not. n = 3. (C) Statistical analysis of bar charts displayed the GFP/RFP-LC3 ratio in MRC-5 cells treated with CQ (50 μM) for 4 h after pre-treatment with FASN shRNA or NC. n = 3. (D) Statistical analysis of bar charts displayed the GFP/RFP-LC3 ratio in MRC-5 cells treated with CQ (50 μM) for 4 h after pre-treatment with C75 or not. n = 3.

Article Snippet: Human FASN shRNA (Targeted 5′-CCTACTGGATGCGTTCTTCAA-3′) and mouse FASN shRNA (Targeted 5′-GCTGGTCGTTTCTCCATTAAA-3′) were designed and synthesized by Sangon Biotech and subsequently annealed and inserted into the pLKO.1.

Techniques: Expressing, shRNA