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Identification of the antitumor targets of ATA. ( A ) Overlap of the predicted targets of ATA (blue) and disease targets from the transcriptomic data (pink). ( B ) Function analysis of fifteen common targets via GeneMANIA. (C) Binding pattern diagram of ATA and the target proteins <t>FDFT1,</t> PPARA, and PPARG. The yellow dashed line represents the hydrogen bond interaction. ( D – F ) After pre-incubation with or without YM-53601 (2.5 μM and 5 µM, ( D )), GW6471 (2.5 μM and 5 µM, ( E )), or GW9662 (5 μM and 10 µM, ( F )) for 30 min, HCT116 cells were treated with ATA (0 μM, 10 μM, and 15 µM) for 48 h. The cell viabilities were detected via the MTT assay. The data are expressed as means ± SD ( n = 3). ** p < 0.01 and *** p < 0.001 vs. control (Ctrl). ( G ) Venn diagram of predicted targets for ATA, OA, and UA. Five genes in red were identified as ATA-specific targets. ( H ) The cell viabilities of HCT116 cells treated with ATA, UA, and OA for 48 h were detected via the MTT assay and the IC 50 values were calculated using Graphpad Prism 9.0 software.

Anti Fdft1 Antibody, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 20243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "3 β -Hydroxy-12-oleanen-27-oic Acid Exerts an Antiproliferative Effect on Human Colon Carcinoma HCT116 Cells via Targeting FDFT1"

Article Title: 3 β -Hydroxy-12-oleanen-27-oic Acid Exerts an Antiproliferative Effect on Human Colon Carcinoma HCT116 Cells via Targeting FDFT1

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms241915020

Figure Legend Snippet: Identification of the antitumor targets of ATA. ( A ) Overlap of the predicted targets of ATA (blue) and disease targets from the transcriptomic data (pink). ( B ) Function analysis of fifteen common targets via GeneMANIA. (C) Binding pattern diagram of ATA and the target proteins FDFT1, PPARA, and PPARG. The yellow dashed line represents the hydrogen bond interaction. ( D – F ) After pre-incubation with or without YM-53601 (2.5 μM and 5 µM, ( D )), GW6471 (2.5 μM and 5 µM, ( E )), or GW9662 (5 μM and 10 µM, ( F )) for 30 min, HCT116 cells were treated with ATA (0 μM, 10 μM, and 15 µM) for 48 h. The cell viabilities were detected via the MTT assay. The data are expressed as means ± SD ( n = 3). ** p < 0.01 and *** p < 0.001 vs. control (Ctrl). ( G ) Venn diagram of predicted targets for ATA, OA, and UA. Five genes in red were identified as ATA-specific targets. ( H ) The cell viabilities of HCT116 cells treated with ATA, UA, and OA for 48 h were detected via the MTT assay and the IC 50 values were calculated using Graphpad Prism 9.0 software.

Techniques Used: Binding Assay, Incubation, MTT Assay, Control, Software

FDFT1 mediated the cytotoxicity of ATA towards HCT116 cells. ( A ) The gene expression levels of FDFT1 in the HCT116 cells treated with ATA for different times via the RT-qPCR assay. ( B , C ) The protein expression levels of FDFT1 in the HCT116 cells treated with ATA (7.5, 15 and 30 μM) for 24 h via Western blotting. The figure ( B ) shown is representative of three independent experiments. The data ( C ) are expressed as means ± SD ( n = 3). * p < 0.05 and ** p < 0.01 vs. Ctrl (0 μM). ( D – F ) After pre-incubation with or without the FDFT1 inhibitor YM-53601 (2 and 4 µM) for 30 min, HCT116 cells were treated with ATA (0 µM, 5 µM, and 7.5 µM, D), OA (100 μM, E), or UA (40 μM, F) for 24 h. The cell viabilities were detected via the MTT assay. The data are expressed as means ± SD ( n = 3). * p < 0.05 vs. the control (Ctrl). ( G – H ) After pre-incubation with or without YM-53601 (4 µM, 30 min), the HCT116 cells were treated with ATA (20 μM) for 24 h. The autophagic flux was observed through the mRFP-GFP-LC3 assay. The figure ( G ) shown is representative of three independent experiments. The cell apoptosis was determined using FCM. The figure ( H ) is representative of three independent experiments. The apoptotic percentages ( I ) are expressed as means ± SD ( n = 3). *** p < 0.001 vs. Ctrl. ( J – L ) The mRNA ( J ) and protein ( K , L ) expression levels of FDFT1 in HCT116 cells were detected using RT-qPCR and Western blotting after transfection with FDFT1 siRNA for 24 h and 48 h, respectively. siNC—negative control siRNA. The figure ( K ) shown is representative of three independent experiments. The data ( J , L ) are expressed as means ± SD ( n = 3). * p < 0.05 and *** p < 0.001 vs. siNC. ( M ) The HCT116 cells were transfected with FDFT1 siRNAs for 48 h, followed by exposure to ATA at indicated concentrations for another 48 h. The cell viabilities were detected through the MTT assay. The data are expressed as means ± SD ( n = 3). * p < 0.05 and *** p < 0.001 vs. siNC.

Figure Legend Snippet: FDFT1 mediated the cytotoxicity of ATA towards HCT116 cells. ( A ) The gene expression levels of FDFT1 in the HCT116 cells treated with ATA for different times via the RT-qPCR assay. ( B , C ) The protein expression levels of FDFT1 in the HCT116 cells treated with ATA (7.5, 15 and 30 μM) for 24 h via Western blotting. The figure ( B ) shown is representative of three independent experiments. The data ( C ) are expressed as means ± SD ( n = 3). * p < 0.05 and ** p < 0.01 vs. Ctrl (0 μM). ( D – F ) After pre-incubation with or without the FDFT1 inhibitor YM-53601 (2 and 4 µM) for 30 min, HCT116 cells were treated with ATA (0 µM, 5 µM, and 7.5 µM, D), OA (100 μM, E), or UA (40 μM, F) for 24 h. The cell viabilities were detected via the MTT assay. The data are expressed as means ± SD ( n = 3). * p < 0.05 vs. the control (Ctrl). ( G – H ) After pre-incubation with or without YM-53601 (4 µM, 30 min), the HCT116 cells were treated with ATA (20 μM) for 24 h. The autophagic flux was observed through the mRFP-GFP-LC3 assay. The figure ( G ) shown is representative of three independent experiments. The cell apoptosis was determined using FCM. The figure ( H ) is representative of three independent experiments. The apoptotic percentages ( I ) are expressed as means ± SD ( n = 3). *** p < 0.001 vs. Ctrl. ( J – L ) The mRNA ( J ) and protein ( K , L ) expression levels of FDFT1 in HCT116 cells were detected using RT-qPCR and Western blotting after transfection with FDFT1 siRNA for 24 h and 48 h, respectively. siNC—negative control siRNA. The figure ( K ) shown is representative of three independent experiments. The data ( J , L ) are expressed as means ± SD ( n = 3). * p < 0.05 and *** p < 0.001 vs. siNC. ( M ) The HCT116 cells were transfected with FDFT1 siRNAs for 48 h, followed by exposure to ATA at indicated concentrations for another 48 h. The cell viabilities were detected through the MTT assay. The data are expressed as means ± SD ( n = 3). * p < 0.05 and *** p < 0.001 vs. siNC.

Techniques Used: Gene Expression, Quantitative RT-PCR, Expressing, Western Blot, Incubation, MTT Assay, Control, Transfection, Negative Control

ATA inhibited the tumor growth of HCT116 xenografts in nude mice. ( A ) Tumor anatomy of HCT116 subcutaneous tumor-bearing mice. Scale bar = 10 mm. ( B ) Growth curve of tumor volume. ( C ) Average final tumor weights of each group. ( D ) Body weights of tumor-bearing mice. ( E , F ) Expression levels of FDFT1, LC3, p62, cyclinD1, pro-caspase3, and GPX4 in tumor tissues from Ctrl and ATA (60 mg/kg) groups via Western blotting. The data were expressed as means ± SD ( n = 4). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Ctrl.

Figure Legend Snippet: ATA inhibited the tumor growth of HCT116 xenografts in nude mice. ( A ) Tumor anatomy of HCT116 subcutaneous tumor-bearing mice. Scale bar = 10 mm. ( B ) Growth curve of tumor volume. ( C ) Average final tumor weights of each group. ( D ) Body weights of tumor-bearing mice. ( E , F ) Expression levels of FDFT1, LC3, p62, cyclinD1, pro-caspase3, and GPX4 in tumor tissues from Ctrl and ATA (60 mg/kg) groups via Western blotting. The data were expressed as means ± SD ( n = 4). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Ctrl.

Techniques Used: Expressing, Western Blot

Figure Legend Snippet: Hypothetical pathway of FDFT1 in mediating the cytotoxic effect of ATA against HCT116 cells.

Techniques Used:

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Article Title: Astrocyte-oligodendrocyte interaction regulates central nervous system regeneration.
Article Snippet: The following antibodies were used: rat antimyelin basic protein (MBP, 1:100, Bio-Rad antibodiesMCA409S), mouse anti-myelin-associated glycoprotein (MAG, 1:100, Sigma-Aldrich MAB1567), chicken anti-glial fibrillary acidic protein (GFAP, 1:500, BioLegend PCK-591P), chicken anti-GFAP (1:500, Cambridge Bioscience 829401), rabbit anti-SOX9 (1:500, Sigma-Aldrich AB5535), mouse antiNestin (1:100, Abcam ab6142), mouse anti-vimentin (1:100, SigmaAldrich clone LN-6 MAB1681), rabbit anti-nuclear factor IA (NFIA, 1:250, Abcam ab228897), rabbit anti-Ki67 (1:500, Sigma-Aldrich AB9260), mouse anti-APC (CC1, 1:100, Abcam ab16794), rabbit anti-Olig2 (1:100, Sigma-Aldrich AB9610), rabbit anti-HMOX1 (1:100, EnzoADI-SPA-895-D), rabbit anti-HMGCS1 (1:500, Invitrogen PA5-29488), rabbit anti-FDPS (1:500, Invitrogen PA5-28228), anti-MVD antibody (1:100, Abcam ab198823) rabbit anti-FDFT1 (1:100, Abcam ab236666), rabbit anti-IBA1 (1:500, Abcam 178846), rabbit anti-ABCA1 (1:100, Novus Biologicals NB400-105), mouse anti-Sox10 (1:100, Sigma-Aldrich AMAB91297), mouse anti-Olig1 (1:1000, EMD Millipore MAB5540), rabbit anti-active Caspase-3 (1:500, BD Pharmingen 559565), mouse anti-CNPase (1:2000, Sigma-Aldrich AMAB91072), mouse anti-MASH1 (1:100, BD Bioscience 556604), rabbit anti-NQO1 (1:100, Abcam ab2346), rat anti-NRF2 (1:500, Cell Signalling 14596S) and chicken anti-GFP (1:100, Abcam ab13970).

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CE inhibits key cholesterol synthesis enzymes and lowers cholesterol levels in SCLC cells. (A) Intersection of the DEGs associated with cholesterol metabolism from RNA-Seq data and 60 predicted target genes identified through network pharmacology. (B) Schematic representation of the cholesterol biosynthesis pathway, highlighting critical rate-limiting enzymes. (C) RT-PCR analysis demonstrating mRNA levels of HMGCS1, HMGCR, IDI1, FDFT1, and SQLE following CE treatment. (D) Western blot analysis showing protein levels of the five genes after CE treatment. (E) Cholesterol content in H1688 cells treated with or without CE (1 µM) for 24 h. (F, G) Representative tumor image (F) and tumor weight (G) of H1688 cells-derived xenografts treated with or without CE (10 mg/kg), n = 3. (H, I) Measurement of cholesterol concentration in serum (H) and tumor tissues (I) of H1688 cells-derived xenografts. (J) The binding energy between corresponding gene and CE. Data are presented as means ± SD of three simultaneously performed experiments (C, E, H, I) . * P < 0.05, ** P < 0.01, *** P < 0.001.

Journal: Frontiers in Pharmacology

Article Title: Cepharanthine as an effective small cell lung cancer inhibitor: integrated insights from network pharmacology, RNA sequencing, and experimental validation

doi: 10.3389/fphar.2024.1517386

Figure Lengend Snippet: CE inhibits key cholesterol synthesis enzymes and lowers cholesterol levels in SCLC cells. (A) Intersection of the DEGs associated with cholesterol metabolism from RNA-Seq data and 60 predicted target genes identified through network pharmacology. (B) Schematic representation of the cholesterol biosynthesis pathway, highlighting critical rate-limiting enzymes. (C) RT-PCR analysis demonstrating mRNA levels of HMGCS1, HMGCR, IDI1, FDFT1, and SQLE following CE treatment. (D) Western blot analysis showing protein levels of the five genes after CE treatment. (E) Cholesterol content in H1688 cells treated with or without CE (1 µM) for 24 h. (F, G) Representative tumor image (F) and tumor weight (G) of H1688 cells-derived xenografts treated with or without CE (10 mg/kg), n = 3. (H, I) Measurement of cholesterol concentration in serum (H) and tumor tissues (I) of H1688 cells-derived xenografts. (J) The binding energy between corresponding gene and CE. Data are presented as means ± SD of three simultaneously performed experiments (C, E, H, I) . * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Membranes were blocked with 5% non-fat dry milk in TBST for 1 h, followed by overnight incubation with primary antibodies: SQLE (A2428), FDFT1 (A4651), HMGCS1 (A3916), and HMGCR (A1633) (Abclonal Technology, Wuhan, China); IDI1 (ab97448) and GAPDH (ab9485) (Abcam, Cambridge, UK).

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, Concentration Assay, Binding Assay

High expression of key cholesterol synthesis enzymes promotes SCLC cell proliferation and correlates with poor prognosis. (A) The expression levels of corresponding gene’s mRNA in SCLC, NSCLC, and other cancer cell lines were obtained from the Cancer Cell Line Encyclopedia (CCLE). (B) Overall survival analysis of human SCLC samples based on the corresponding gene expression. (C) RT-PCR was used to analyze the expression levels of key cholesterol synthesis enzymes (HMGCS1, HMGCR, IDI1, FDFT1, and SQLE) in 16HBE (human bronchial epithelial cell) and SCLC cell lines. (D) Protein expression analysis was conducted on the corresponding genes in H1688 cells transfected with either shCtl or shRNA targeting each specific gene. (E) Cell proliferation was measured after silencing of HMGCS1, HMGCR, IDI1, FDFT1, and SQLE in H1688 cells. Data are presented as means ± SD of three simultaneously performed experiments (C, E) . * P < 0.05, ** P < 0.01, *** P < 0.001.

Journal: Frontiers in Pharmacology

Article Title: Cepharanthine as an effective small cell lung cancer inhibitor: integrated insights from network pharmacology, RNA sequencing, and experimental validation

doi: 10.3389/fphar.2024.1517386

Figure Lengend Snippet: High expression of key cholesterol synthesis enzymes promotes SCLC cell proliferation and correlates with poor prognosis. (A) The expression levels of corresponding gene’s mRNA in SCLC, NSCLC, and other cancer cell lines were obtained from the Cancer Cell Line Encyclopedia (CCLE). (B) Overall survival analysis of human SCLC samples based on the corresponding gene expression. (C) RT-PCR was used to analyze the expression levels of key cholesterol synthesis enzymes (HMGCS1, HMGCR, IDI1, FDFT1, and SQLE) in 16HBE (human bronchial epithelial cell) and SCLC cell lines. (D) Protein expression analysis was conducted on the corresponding genes in H1688 cells transfected with either shCtl or shRNA targeting each specific gene. (E) Cell proliferation was measured after silencing of HMGCS1, HMGCR, IDI1, FDFT1, and SQLE in H1688 cells. Data are presented as means ± SD of three simultaneously performed experiments (C, E) . * P < 0.05, ** P < 0.01, *** P < 0.001.

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA

Journal: Journal of Dental Sciences

Article Title: Periodontal ligament fibroblasts utilize isoprenoid intermediate farnesyl diphosphate for maintaining osteo/cementogenic differentiation abilities

doi: 10.1016/j.jds.2024.04.025

Figure Lengend Snippet: Primer pairs used in this study.

Article Snippet: The sections were incubated with rabbit IgG (DA1E, Cell Signaling Technologies, Danvers, MA, final concentration 10 μg/mL), rabbit anti-farnesyl diphosphate farnesyltransferase 1 (FDFT1) (13128-1-AP, Proteintech, Rosemont, IL, final concentration 10 μg/mL), rabbit anti-farnesyl pyrophosphate synthetase (FDPS) (16129-1-AP, Proteintech, final concentration 10 μg/mL), and rabbit anti-geranylgeranyl diphosphate synthase 1 (GGPS1) (14944-1-AP, Proteintech, final concentration 10 μg/mL) at 4 °C overnight.

Techniques: Sequencing

The isoprenoid metabolic process is most severely downregulated metabolic pathway in PPARγ-knockdown PDLFs. (A) Gene ontology analysis of the downregulated genes in PDLFs transfected with siRNA for PPARG by comparing with PDLFs transfected with control siRNA. Raw RNA-seq data were obtained from NCBI's Gene Expression Omnibus (GEO) under accession number GSE178607 . (B) PDLF-1 cells were cultured in mineralization-inducing medium for 3 and 6 days in the presence of troglitazone (10 μM) or rosiglitazone (10 μM). The gene expression levels of AKR1C3 , ALDH3A2 , AKR1C1 , and SDC3 , which were categorized in “isoprenoid metabolic process” and identified as the downregulated genes in PPARG-knockdown PDLFs, were evaluated. (C) PDLFs were cultured in mineralization-inducing medium for 6 days in the presence of troglitazone (10 μM) or rosiglitazone (10 μM) and FPP (20 μM) or GGPP (20 μM). ALP activities were normalized by cell numbers. (D) PDLFs were cultured in mineralization-inducing medium for 12 days in the presence of FPP (0, 10, 20 μM), and calcium deposition was visualized and quantified by Alizarin Red S staining. (E) Demineralized 1.5-month-old maxilla sections were stained without the primary antibody or with mouse IgG, α-FDPS, α-GGPS1, or α-FDFT1 antibodies, ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 indicated significantly higher expression levels compared with those in DMSO-treated PDLFs at each day point (B), non-treated PDLFs in either DMSO, troglitazone, or rosiglitazone treatment group (C), and non-treated PDLFs (D). Tro = troglitazone. Ros = rosiglitazone. P = pulp. D = dentin. Pdl = periodontal ligament tissue. Ab = alveolar bone. Scale bars correspond to 100 μm.

Journal: Journal of Dental Sciences

Article Title: Periodontal ligament fibroblasts utilize isoprenoid intermediate farnesyl diphosphate for maintaining osteo/cementogenic differentiation abilities

doi: 10.1016/j.jds.2024.04.025

Figure Lengend Snippet: The isoprenoid metabolic process is most severely downregulated metabolic pathway in PPARγ-knockdown PDLFs. (A) Gene ontology analysis of the downregulated genes in PDLFs transfected with siRNA for PPARG by comparing with PDLFs transfected with control siRNA. Raw RNA-seq data were obtained from NCBI's Gene Expression Omnibus (GEO) under accession number GSE178607 . (B) PDLF-1 cells were cultured in mineralization-inducing medium for 3 and 6 days in the presence of troglitazone (10 μM) or rosiglitazone (10 μM). The gene expression levels of AKR1C3 , ALDH3A2 , AKR1C1 , and SDC3 , which were categorized in “isoprenoid metabolic process” and identified as the downregulated genes in PPARG-knockdown PDLFs, were evaluated. (C) PDLFs were cultured in mineralization-inducing medium for 6 days in the presence of troglitazone (10 μM) or rosiglitazone (10 μM) and FPP (20 μM) or GGPP (20 μM). ALP activities were normalized by cell numbers. (D) PDLFs were cultured in mineralization-inducing medium for 12 days in the presence of FPP (0, 10, 20 μM), and calcium deposition was visualized and quantified by Alizarin Red S staining. (E) Demineralized 1.5-month-old maxilla sections were stained without the primary antibody or with mouse IgG, α-FDPS, α-GGPS1, or α-FDFT1 antibodies, ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 indicated significantly higher expression levels compared with those in DMSO-treated PDLFs at each day point (B), non-treated PDLFs in either DMSO, troglitazone, or rosiglitazone treatment group (C), and non-treated PDLFs (D). Tro = troglitazone. Ros = rosiglitazone. P = pulp. D = dentin. Pdl = periodontal ligament tissue. Ab = alveolar bone. Scale bars correspond to 100 μm.

Techniques: Knockdown, Transfection, Control, RNA Sequencing, Gene Expression, Cell Culture, Staining, Expressing

Correlation between OCN or COL1A1 and FDPS , GGPS1 , or FDFT1 expression in human clinical periodontal tissue. Human clinical periodontal tissues (18 samples from different patients) were collected, and the correlative expression levels of OCN or COL1A1 with FDPS , GGPS1, or FDFT1 were examined. The high (r > 0.8 or r < −0.8) and weak correlations (0.2 < r < 0.4 or −0.4 < r < −0.2) are indicated by green and orange lines, respectively.

Journal: Journal of Dental Sciences

Article Title: Periodontal ligament fibroblasts utilize isoprenoid intermediate farnesyl diphosphate for maintaining osteo/cementogenic differentiation abilities

doi: 10.1016/j.jds.2024.04.025

Figure Lengend Snippet: Correlation between OCN or COL1A1 and FDPS , GGPS1 , or FDFT1 expression in human clinical periodontal tissue. Human clinical periodontal tissues (18 samples from different patients) were collected, and the correlative expression levels of OCN or COL1A1 with FDPS , GGPS1, or FDFT1 were examined. The high (r > 0.8 or r < −0.8) and weak correlations (0.2 < r < 0.4 or −0.4 < r < −0.2) are indicated by green and orange lines, respectively.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: 3 β -Hydroxy-12-oleanen-27-oic Acid Exerts an Antiproliferative Effect on Human Colon Carcinoma HCT116 Cells via Targeting FDFT1

doi: 10.3390/ijms241915020

Figure Lengend Snippet: Identification of the antitumor targets of ATA. ( A ) Overlap of the predicted targets of ATA (blue) and disease targets from the transcriptomic data (pink). ( B ) Function analysis of fifteen common targets via GeneMANIA. (C) Binding pattern diagram of ATA and the target proteins FDFT1, PPARA, and PPARG. The yellow dashed line represents the hydrogen bond interaction. ( D – F ) After pre-incubation with or without YM-53601 (2.5 μM and 5 µM, ( D )), GW6471 (2.5 μM and 5 µM, ( E )), or GW9662 (5 μM and 10 µM, ( F )) for 30 min, HCT116 cells were treated with ATA (0 μM, 10 μM, and 15 µM) for 48 h. The cell viabilities were detected via the MTT assay. The data are expressed as means ± SD ( n = 3). ** p < 0.01 and *** p < 0.001 vs. control (Ctrl). ( G ) Venn diagram of predicted targets for ATA, OA, and UA. Five genes in red were identified as ATA-specific targets. ( H ) The cell viabilities of HCT116 cells treated with ATA, UA, and OA for 48 h were detected via the MTT assay and the IC 50 values were calculated using Graphpad Prism 9.0 software.

Article Snippet: For this study, 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), acridine orange (AO), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, St. Louis, MO, USA; cell culture medium, trypsin, penicillin, streptomycin, and fetal bovine serum (FBS) were obtained from Gibco, Burlington, MA, USA; TRIzol was purchased from Invitrogen, Carlsbad, CA, USA; reverse transcriptase, oligo(dT) 18 , and ribonuclease inhibitor were obtained from Shanghai Sangon Biotech Co., Ltd., Shanghai, China; RIPA lysis buffer, BCA protein assay kit, anti-mouse actin mAbs, anti-caspase3 antibody (AF0081), horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG (H + L), enhanced chemiluminescence (ECL) kit, and reactive oxygen species (ROS) assay kit were purchased from Beyotime, Shanghai, China; phosphatase inhibitor and protease inhibitor cocktails were obtained from Biotool, Houston, TX, USA; anti-FDFT1 antibody (ab195046) was purchased from Abcam, Cambridge, UK; anti-cyclinD1 antibody (ET1601-31) was obtained from HUABIO, Shanghai, China; anti-GPX4 (67763-1-Ig) antibody was obtained from Proteintech, Rosemont, IL, USA; the antibodies against LC3A/B (4108S) and p62 (39749S) were purchased from CST, Danvers, MA, USA; a monodansylcadaverine (MDC) sensor kit was obtained from KeyGEN BioTECH Corp., Ltd., Nanjing, China; an annexin V-FITC apoptosis detection kit and propidium iodide (PI)/RNase staining buffer were obtained from BD Pharmingen, San Diego, CA, USA; mRFP-GFP-LC3 lentivirus was obtained from Genechem, Shanghai, China.

Techniques: Binding Assay, Incubation, MTT Assay, Control, Software

Journal: International Journal of Molecular Sciences

Article Title: 3 β -Hydroxy-12-oleanen-27-oic Acid Exerts an Antiproliferative Effect on Human Colon Carcinoma HCT116 Cells via Targeting FDFT1

doi: 10.3390/ijms241915020

Figure Lengend Snippet: FDFT1 mediated the cytotoxicity of ATA towards HCT116 cells. ( A ) The gene expression levels of FDFT1 in the HCT116 cells treated with ATA for different times via the RT-qPCR assay. ( B , C ) The protein expression levels of FDFT1 in the HCT116 cells treated with ATA (7.5, 15 and 30 μM) for 24 h via Western blotting. The figure ( B ) shown is representative of three independent experiments. The data ( C ) are expressed as means ± SD ( n = 3). * p < 0.05 and ** p < 0.01 vs. Ctrl (0 μM). ( D – F ) After pre-incubation with or without the FDFT1 inhibitor YM-53601 (2 and 4 µM) for 30 min, HCT116 cells were treated with ATA (0 µM, 5 µM, and 7.5 µM, D), OA (100 μM, E), or UA (40 μM, F) for 24 h. The cell viabilities were detected via the MTT assay. The data are expressed as means ± SD ( n = 3). * p < 0.05 vs. the control (Ctrl). ( G – H ) After pre-incubation with or without YM-53601 (4 µM, 30 min), the HCT116 cells were treated with ATA (20 μM) for 24 h. The autophagic flux was observed through the mRFP-GFP-LC3 assay. The figure ( G ) shown is representative of three independent experiments. The cell apoptosis was determined using FCM. The figure ( H ) is representative of three independent experiments. The apoptotic percentages ( I ) are expressed as means ± SD ( n = 3). *** p < 0.001 vs. Ctrl. ( J – L ) The mRNA ( J ) and protein ( K , L ) expression levels of FDFT1 in HCT116 cells were detected using RT-qPCR and Western blotting after transfection with FDFT1 siRNA for 24 h and 48 h, respectively. siNC—negative control siRNA. The figure ( K ) shown is representative of three independent experiments. The data ( J , L ) are expressed as means ± SD ( n = 3). * p < 0.05 and *** p < 0.001 vs. siNC. ( M ) The HCT116 cells were transfected with FDFT1 siRNAs for 48 h, followed by exposure to ATA at indicated concentrations for another 48 h. The cell viabilities were detected through the MTT assay. The data are expressed as means ± SD ( n = 3). * p < 0.05 and *** p < 0.001 vs. siNC.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Western Blot, Incubation, MTT Assay, Control, Transfection, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: 3 β -Hydroxy-12-oleanen-27-oic Acid Exerts an Antiproliferative Effect on Human Colon Carcinoma HCT116 Cells via Targeting FDFT1

doi: 10.3390/ijms241915020

Figure Lengend Snippet: ATA inhibited the tumor growth of HCT116 xenografts in nude mice. ( A ) Tumor anatomy of HCT116 subcutaneous tumor-bearing mice. Scale bar = 10 mm. ( B ) Growth curve of tumor volume. ( C ) Average final tumor weights of each group. ( D ) Body weights of tumor-bearing mice. ( E , F ) Expression levels of FDFT1, LC3, p62, cyclinD1, pro-caspase3, and GPX4 in tumor tissues from Ctrl and ATA (60 mg/kg) groups via Western blotting. The data were expressed as means ± SD ( n = 4). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Ctrl.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: 3 β -Hydroxy-12-oleanen-27-oic Acid Exerts an Antiproliferative Effect on Human Colon Carcinoma HCT116 Cells via Targeting FDFT1

doi: 10.3390/ijms241915020

Figure Lengend Snippet: Hypothetical pathway of FDFT1 in mediating the cytotoxic effect of ATA against HCT116 cells.

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