Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Ehrlichia chaffeensis Tandem Repeat Effector Targets Differentially Influence Infection

doi: 10.3389/fcimb.2017.00178

Figure Lengend Snippet: E. chaffeensis TRP-interacting host proteins involved in cell apoptosis .

Article Snippet: Other antibodies used in this study were mouse anti-human FBXW7 (R&D Systems, Minneapolis, MN), α-tubulin and PTPN2 (Santa Cruz, Dallas, TX), and rabbit anti-human CD14 (Abgent, San Diego, CA), EIF3A, ERA1, KARS, LMAN1, and STAT6 (Proteintech, Rosemont, IL), FYN and SLC43A3 (Santa Cruz), IRF2BP2 and SEPX1 (Pierce, Rockford, IL), KDM6B (Novus, Littleton, CO), and RC3H1 (Sigma).

Techniques: Binding Assay, Modification, Histone Deacetylase Assay

Journal: Nature

Article Title: PVT1 dependence in cancer with MYC copy-number increase

doi: 10.1038/nature13311

Figure Lengend Snippet: a, RT–qPCR measurement of Myc (left) and Pvt1 (right) RNA levels in gain(Myc, Pvt1, Ccdc26, Gsdmc), MMTVneu/+ mammary tumour cells transfected with indicated siRNAs (n = 3). b, Proportions of primary tumour cells positive for Ki-67 after the indicated siRNA treatments (n = 3). c, RT–qPCR of Myc (left) and Pvt1 (right) transcript levels (n = 3), and d, western blot analysis (top) and quantification (bottom) of Myc protein in mammary tissue (n = 3). GAPDH, Glyceraldehyde 3-phosphate dehydrogenase. e–h, Analyses of human breast cancer cell line SK-BR-3. e, RT–qPCR measurement of MYC (left) and PVT1 (right) transcripts in cells 48 h after transfection with the indicated siRNA(s) (n = 3). f, g, Western blot analysis of the MYC protein in SK-BR-3 after siRNA transfection (n = 3) (f), and siRNA transfection and cycloheximide (CHX) treatment for times indicated (n = 3) (g). h, Western blot analysis of MYC(p-T58), MYC(p-S62), MYC, FBW7 and AXIN1 protein levels in SK-BR-3 treated with siRNAs (left). Ratios of T58/total MYC and p-S62/total MYC (right) (n = 3). i, Immunofluorescence staining of MYC (green) and RNA FISH of PVT1 (magenta) showing nuclear co-localization of MYC and PVT1 (white). 4′,6-Diamidino-2-phenylindole (DAPI) is shown in blue. The marked cell in the upper panel is shown in the lower panels in single channels and MYC + PVT1 overlay. j, RT–PCR using PVT1 and GAPDH specific primers of total SK-BR-3 RNA (input), immunoprecipitated using MYC antibody (IP MYC) and IgG (IP IgG). PVT1–RT indicates samples not treated with reverse transcriptase. Results are shown as mean ± s.e.m. (*P<0.05, **P<0.01, ***P<0.001, two-tailed Student’s t-test). Scale bar, 10 μm; error bars, s.e.m.

Article Snippet: Immunoblotting was done using the following: c-Myc (Abcam, ab32), T58 (Abcam, ab85380), S62 (Abcam, ab51156), FBW7 (Novus Biologicals NBP1-59631), AXIN1 (Cell Signaling, 2087), GAPDH (Cell Signaling, 5174), pERK1/2 (Cell Signaling, 4370), ERK1/2 (Cell Signaling,4695) and P53(Cell Signaling, 2524). siRNAtransfections and three-dimensional culture siRNA against human PVT1 and MYC as well as mouse Pvt1 and Myc were designed and purchased from FlexiTube siRNA (Qiagen).

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Reverse Transcription, Two Tailed Test

Journal: Frontiers in Medicine

Article Title: Whole exome sequencing identifies a novel mutation in Annexin A4 that is associated with recurrent spontaneous abortion

doi: 10.3389/fmed.2024.1462649

Figure Lengend Snippet: Representative sequencing electropherogram of the ANXA4 variant (p.G8D). The arrow indicates the location of the mutation, and patient “RSA-219” harbored the ANXA4 p.G8D variant (A) . Evolutionary conservation analysis of ANXA4 p.G8D in vertebrate species (B) .

Article Snippet: The membranes were subsequently incubated with the following primary antibodies at 4°C overnight: anti- ANXA4 (1:1000, TA803051; Origene, China) and anti- β -actin (1,2000, sc-47778; Santa Cruz).

Techniques: Sequencing, Variant Assay, Mutagenesis

Journal: Frontiers in Medicine

Article Title: Whole exome sequencing identifies a novel mutation in Annexin A4 that is associated with recurrent spontaneous abortion

doi: 10.3389/fmed.2024.1462649

Figure Lengend Snippet: Structural differences between wild-type ANXA4 (p.G8) and mutated ANXA4 (p.D8) proteins. The protein structures of the wild-type and mutated ANXA4 proteins were modeled based on the crystal model of the human ANXA4 protein.

Article Snippet: The membranes were subsequently incubated with the following primary antibodies at 4°C overnight: anti- ANXA4 (1:1000, TA803051; Origene, China) and anti- β -actin (1,2000, sc-47778; Santa Cruz).

Techniques:

Journal: Frontiers in Medicine

Article Title: Whole exome sequencing identifies a novel mutation in Annexin A4 that is associated with recurrent spontaneous abortion

doi: 10.3389/fmed.2024.1462649

Figure Lengend Snippet: The ANXA4 p.G8D mutation inhibited cell migration, invasion and adhesion in stably transfected THESCs. THESCs were cultured and transfected with ANXA4 wild-type (WT) or mutant-type (MT) plasmids, while pcDNA3.1 was used as a control plasmid (Con). Twenty-four hours after transfection, G418 was used to screen stably transfected cells, and one individual clone was separated by dilution. ANXA4 expression in stably transfected cells was confirmed by WB (A,B) and RT–qPCR (C) . The effects of ANXA4 mutation on cell migration (D,F) and invasion (E,G) were determined by Transwell assays, and cell adhesion (H,I) was determined by Matrigel adhesion assays. These assays revealed that ANXA4 p.G8D mutation inhibited cell migration, invasion and adhesion in stably transfected cells compared with ANXA4 WT. Scale bars, 100 μm. The data are shown as the means ± SDs. p < 0.05 was considered to indicate a significant difference.

Article Snippet: The membranes were subsequently incubated with the following primary antibodies at 4°C overnight: anti- ANXA4 (1:1000, TA803051; Origene, China) and anti- β -actin (1,2000, sc-47778; Santa Cruz).

Techniques: Mutagenesis, Migration, Stable Transfection, Transfection, Cell Culture, Control, Plasmid Preparation, Expressing, Quantitative RT-PCR

Journal: STAR Protocols

Article Title: Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9

doi: 10.1016/j.xpro.2022.101978

Figure Lengend Snippet:

Article Snippet: FBXW7/Cdc4 Antibody (1:100) , Novus Biologicals , Cat# H00055294-M02.

Techniques: Recombinant, Software, Cell Culture, Transferring, Membrane, Microscopy, Transfection

Journal: Cancers

Article Title: F-Box/WD Repeat Domain-Containing 7 Induces Chemotherapy Resistance in Colorectal Cancer Stem Cells

doi: 10.3390/cancers11050635

Figure Lengend Snippet: High FBXW7 expression in pre-treatment biopsy specimens is related to poor pathological therapeutic effect. ( A ) IHC staining for FBXW7 in representative pre-treatment biopsy specimens. Left panel shows high FBXW7 expression and right panel shows low FBXW7 expression. Scale bars, 100 µm. ( B ) Correlation between FBXW7 expression in pre-treatment biopsy specimens and the pathological therapeutic effect of NAC/NACRT in surgically resected specimens. X-axis, number of cases.

Article Snippet: Cells were cultured in high glucose DMEM (Wako, Tokyo, Japan) with 10% fetal bovine serum (Life Technologies Japan, Tokyo, Japan) and penicillin (100 U/mL) and streptomycin (100 μg/mL; Life Technologies Japan), and were incubated at 37 °C in a humidified chamber containing 5% CO 2 . shRNA-expressing lentiviruses were produced by transfecting HEK293T cells with human FBXW7 shRNA constructs in lentiviral GFP vectors (TL304541, OriGene Technologies, Rockville, MD, USA) together with the packaging vector psPAX2 and the envelope vector pMD2.G using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Immunohistochemistry

Journal: Cancers

Article Title: F-Box/WD Repeat Domain-Containing 7 Induces Chemotherapy Resistance in Colorectal Cancer Stem Cells

doi: 10.3390/cancers11050635

Figure Lengend Snippet: Correlation between FBXW7 expression and clinicopathological features in 55 patients with CRC treated with NAC/NACRT before surgical resection.

Article Snippet: Cells were cultured in high glucose DMEM (Wako, Tokyo, Japan) with 10% fetal bovine serum (Life Technologies Japan, Tokyo, Japan) and penicillin (100 U/mL) and streptomycin (100 μg/mL; Life Technologies Japan), and were incubated at 37 °C in a humidified chamber containing 5% CO 2 . shRNA-expressing lentiviruses were produced by transfecting HEK293T cells with human FBXW7 shRNA constructs in lentiviral GFP vectors (TL304541, OriGene Technologies, Rockville, MD, USA) together with the packaging vector psPAX2 and the envelope vector pMD2.G using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing

Journal: Cancers

Article Title: F-Box/WD Repeat Domain-Containing 7 Induces Chemotherapy Resistance in Colorectal Cancer Stem Cells

doi: 10.3390/cancers11050635

Figure Lengend Snippet: FBXW7 expression in the post-treatment resected specimens is higher than that in the pre-treatment biopsy specimens. ( A ) Hematoxylin and eosin (H.E.) and IHC staining for FBXW7 in serial sections of representative CRC specimens. Patient #1 belongs to the FBXW7-high group and shows low pathological therapeutic effect. Patient #2 belongs to the FBXW7-low group and shows high pathological therapeutic effect. Scale bars, 100 µm. ( B ) Comparison of FBXW7 staining score between pre-treatment biopsy specimens and post-treatment resected specimens. We evaluated 49 cases in which FBXW7 IHC staining could be performed on both biopsy and surgically resected specimens. ( C ) IHC staining for FBXW7 and Ki-67 in serial sections of representative CRC specimens. Patient #3 belongs to the FBXW7-high group and shows low pathological therapeutic effect. Patient #4 belongs to the FBXW7-low group and shows high pathological therapeutic effect. Scale bars, 100 µm.

Article Snippet: Cells were cultured in high glucose DMEM (Wako, Tokyo, Japan) with 10% fetal bovine serum (Life Technologies Japan, Tokyo, Japan) and penicillin (100 U/mL) and streptomycin (100 μg/mL; Life Technologies Japan), and were incubated at 37 °C in a humidified chamber containing 5% CO 2 . shRNA-expressing lentiviruses were produced by transfecting HEK293T cells with human FBXW7 shRNA constructs in lentiviral GFP vectors (TL304541, OriGene Technologies, Rockville, MD, USA) together with the packaging vector psPAX2 and the envelope vector pMD2.G using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Immunohistochemistry, Comparison, Staining

Journal: Cancers

Article Title: F-Box/WD Repeat Domain-Containing 7 Induces Chemotherapy Resistance in Colorectal Cancer Stem Cells

doi: 10.3390/cancers11050635

Figure Lengend Snippet: FBXW7 staining scores in pre-treatment biopsy specimens and post-treatment resected specimens for each patient.

Article Snippet: Cells were cultured in high glucose DMEM (Wako, Tokyo, Japan) with 10% fetal bovine serum (Life Technologies Japan, Tokyo, Japan) and penicillin (100 U/mL) and streptomycin (100 μg/mL; Life Technologies Japan), and were incubated at 37 °C in a humidified chamber containing 5% CO 2 . shRNA-expressing lentiviruses were produced by transfecting HEK293T cells with human FBXW7 shRNA constructs in lentiviral GFP vectors (TL304541, OriGene Technologies, Rockville, MD, USA) together with the packaging vector psPAX2 and the envelope vector pMD2.G using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Staining

Journal: Cancers

Article Title: F-Box/WD Repeat Domain-Containing 7 Induces Chemotherapy Resistance in Colorectal Cancer Stem Cells

doi: 10.3390/cancers11050635

Figure Lengend Snippet: Analysis of human CRC PDXs. ( A ) Representative flow cytometric plot. The EpCAM high /CD44 high and EpCAM high /CD44 low populations were collected by flow cytometry. ( B ) FBXW7 expression in the four types of PDXs. FBXW7 expression in the EpCAM high /CD44 high population was significantly higher than that in the EpCAM high /CD44 low population in all four PDXs. Results are presented as the means ± standard deviation of at least three independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Cells were cultured in high glucose DMEM (Wako, Tokyo, Japan) with 10% fetal bovine serum (Life Technologies Japan, Tokyo, Japan) and penicillin (100 U/mL) and streptomycin (100 μg/mL; Life Technologies Japan), and were incubated at 37 °C in a humidified chamber containing 5% CO 2 . shRNA-expressing lentiviruses were produced by transfecting HEK293T cells with human FBXW7 shRNA constructs in lentiviral GFP vectors (TL304541, OriGene Technologies, Rockville, MD, USA) together with the packaging vector psPAX2 and the envelope vector pMD2.G using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Flow Cytometry, Expressing, Standard Deviation

Journal: Cancers

Article Title: F-Box/WD Repeat Domain-Containing 7 Induces Chemotherapy Resistance in Colorectal Cancer Stem Cells

doi: 10.3390/cancers11050635

Figure Lengend Snippet: FBXW7 knockdown accelerates cell cycle and cell proliferation and increases sensitivity to anti-cancer drugs in CRC cell lines. ( A ) Cell cycle distributions in control and FBXW7 -silenced cells following release from cell synchronization by double thymidine block. Upper and lower panels for each CRC cell line show cell cycle distribution at 0 and 24 h after release from double thymidine synchronization. Bar graphs indicate the percentage of cells in G0/G1, S or G2/M phase 24 h after release from cell synchronization. ( B ) Luminescence-based cell proliferation assays in luciferase-expressing CRC cell lines in which FBXW7 was stably knocked down. ( C ) Luminescence-based cytotoxicity assays in luciferase-expressing CRC cell lines in which FBXW7 was stably knocked down. Cells were incubated for 24, 48 or 72 h with the indicated concentrations of anti-cancer drugs. Cell viability was calculated as the treated/control cell ratio of photon counts. Results are presented as the means ± standard deviation of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were cultured in high glucose DMEM (Wako, Tokyo, Japan) with 10% fetal bovine serum (Life Technologies Japan, Tokyo, Japan) and penicillin (100 U/mL) and streptomycin (100 μg/mL; Life Technologies Japan), and were incubated at 37 °C in a humidified chamber containing 5% CO 2 . shRNA-expressing lentiviruses were produced by transfecting HEK293T cells with human FBXW7 shRNA constructs in lentiviral GFP vectors (TL304541, OriGene Technologies, Rockville, MD, USA) together with the packaging vector psPAX2 and the envelope vector pMD2.G using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Knockdown, Control, Blocking Assay, Luciferase, Expressing, Stable Transfection, Incubation, Standard Deviation

Journal: Cancers

Article Title: F-Box/WD Repeat Domain-Containing 7 Induces Chemotherapy Resistance in Colorectal Cancer Stem Cells

doi: 10.3390/cancers11050635

Figure Lengend Snippet: In a xenograft model, FBXW7 knockdown promotes tumor growth and induces tumor inhibition upon treatment with CPT-11. ( A ) Representative images of tumors in nude mice inoculated with control or FBXW7-silenced DLD-1 cells, treated or not with CPT-11, 31 days after inoculation. White scale bars, 1 cm. ( B ) Macroscopic images of the tumors in nude mice inoculated with control or FBXW7 -silenced DLD-1 cells, treated or not with CPT-11, 31 days after inoculation. ( C ) Tumor growth curves of xenografts derived mice inoculated with control or FBXW7 -silenced DLD-1 cells, treated or not with CPT-11. (D) Tumor growth inhibition rates. Results are presented as the means ± standard deviation of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were cultured in high glucose DMEM (Wako, Tokyo, Japan) with 10% fetal bovine serum (Life Technologies Japan, Tokyo, Japan) and penicillin (100 U/mL) and streptomycin (100 μg/mL; Life Technologies Japan), and were incubated at 37 °C in a humidified chamber containing 5% CO 2 . shRNA-expressing lentiviruses were produced by transfecting HEK293T cells with human FBXW7 shRNA constructs in lentiviral GFP vectors (TL304541, OriGene Technologies, Rockville, MD, USA) together with the packaging vector psPAX2 and the envelope vector pMD2.G using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Knockdown, Inhibition, Control, Derivative Assay, Standard Deviation