Journal: International Journal of Biological Sciences

Article Title: Alcohol Increases Liver Progenitor Populations and Induces Disease Phenotypes in Human IPSC-Derived Mature Stage Hepatic Cells

doi: 10.7150/ijbs.15811

Figure Lengend Snippet: Alcohol increases disease phenotypes including lipid accumulation and hepatocellular carcinoma markers, and oxidative mitochondrial injury in human iPSC-derived mature stage hepatocytes. (A) Lipid droplets were stained with Oil Red O in control groups and ethanol treated groups. (B) After staining, Oil Red O was extracted by isopropanol and the absorbance at 492nm was measured. (C) Expression levels of fatty acid synthase (FASN), hepatocellular carcinoma markers glypican3 (GPC3) and filamin B (FLNB), and tumor suppressor, TP53 were measured by Real-time PCR. (D) Immunostaining of 8-OHdG, a marker for oxidative mitochondrial DNA damage, in alcohol treated day 25 mature stage hepatocytes derived from human iPSCs. (E) Expression of IL-6 and Neil 1 were examined by Real-time PCR in the day 25 hepatocytes exposed to alcohol 0 to 200 mM. (F) NAC treatment restored expression of Neil-1, FASN, and GPC3 in the alcohol treated mature hepatocytes. The treatment period and cell stage for Figure is the same as those used for Figure . *: p <0.05, Scale Bar, 100µm.

Article Snippet: PCRs were performed with 20 ng of complementary DNA, TaqMan Fast Advanced Master Mix, and TaqMan gene expression assay reagent for: AFP (Hs00173490_m1), ALB (Hs00910225_m1), CK7 (Hs00559840_m1), IL-6 (Hs00985639_m1), CK18 (Hs02827483_g1), CK19 (Hs00761767_s1), FASN (Hs01005622_m1), SOX17 (Hs00751752_s1), GPC3 (Hs01018936_m1), FLNB (Hs00181698_m1), CYP2E1 (Hs00559368_m1), CD133 (Hs01009250_m1), EpCAM (Hs00901885_m1), CYP3A4 (Hs00604506_m1), TP53(Hs01034249_m1), Neil1(Hs00908563_m1) and 18S rRNA (Hs03003631_g1) in a total volume of 20 ul.

Techniques: Derivative Assay, Staining, Control, Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Marker

Journal: Reproductive toxicology (Elmsford, N.Y.)

Article Title: Determinants of Orofacial Clefting I: Effects of 5-Aza-2′-deoxycytidine on Cellular Processes and Gene Expression during Development of the First Branchial Arch

doi: 10.1016/j.reprotox.2016.11.016

Figure Lengend Snippet: Genes Differentially Expressed within 1 st Branchial Arches Following 6 hour in utero Exposure to 1 mg/kg AzaD on GD 9.5.

Article Snippet: Flnb , Filamin B, beta , Mm01311723_m1 , −2.13 , 0.04.

Techniques: In Utero, TaqMan Assay, Binding Assay, Membrane, Ubiquitin Proteomics, Histone Deacetylase Assay

Journal: Oncotarget

Article Title: Filamin C promotes lymphatic invasion and lymphatic metastasis and increases cell motility by regulating Rho GTPase in esophageal squamous cell carcinoma

doi: 10.18632/oncotarget.14087

Figure Lengend Snippet: ( A ) Infection efficiency of shRNA lentiviral vectors in the ESCC cell lines (TE-1 and TE-8). Infection efficiency of shRNA lentiviral vectors was confirmed by visualization of green fluorescent protein (GFP) with a confocal microscope. Nuclei are stained with DAPI to visualize the cells. More than 90% of cells were confirmed to be infected with shRNA lentivirus. ( B , C ) mRNA and protein expression levels of FLNA, FLNB, and FLNC were examined by real-time PCR and immunoblotting. GAPDH was used as a loading control. The FLNA and FLNB expression were not reduced in FLNC shRNA infected cells, and were nearly identical to those of SshRNA infected cells. FLNC expression was clearly inhibited in FLNC shRNA infected cells as compared with its expression in SshRNA infected cells. Densities of the immunoblot bands were quantified using Image J software and normalized to GAPDH to obtain the relative densities (RD).

Article Snippet: The following reagents were purchased from the indicated manufacturers: RPMI 1640 (Nikken Biomedical Laboratory, Osaka, Japan); fetal calf serum (FCS) (PAA Laboratories, Pasching, Austria); MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA); DAPI (4′,6-Diamidino-2-phenylindole, dihydrochloride, solution) (Dojindo Laboratories, Kumamoto, Japan); monoclonal antibodies against FLNA, GAPDH (EMD Millipore, Billerica, MA, USA; Cell Signaling Technology, Danvers, MA, USA, respectively), polyclonal antibodies against FLNB, FLNC (EMD Millipore, Billerica, MA, USA; Atlas Antibodies, Stockholm, Sweden, respectively).

Techniques: Infection, shRNA, Microscopy, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Software

Journal: Oncology Reports

Article Title: Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells

doi: 10.3892/or.2020.7532

Figure Lengend Snippet: Knockdown of FLNB promotes proliferation and inhibits apoptosis of HeLa cells. (A) Relative mRNA expression of FLNB in HeLa cells after it was knocked down using FLNB -specific shRNA was determined by reverse transcription-qPCR. shFLNB_1 and shFLNB_2 indicate two biological repeats of HeLa cells transfected with FLNB-specific shRNA. Ctrl_1 and Ctrl_2 indicate two biological repeats of HeLa cells transfected with empty vector as controls. (B) Cell proliferation of shFLNB was measured by an MTT assay in HeLa cells. Cell apoptosis of cells transfected with shFLNB was measured by (C) flow cytometry and (D) subsequent analysis by a 7-ADD and Annexin V assay. *P<0.05 and **P<0.01 vs. respective Ctrl. FLNB , filamin B; sh, short hairpin; qPCR, quantitative PCR; Ctrl, control; 7-AAD, 7-amino actinomycin D; OD, optical density; PE, phycoerythrin.

Article Snippet: Subsequently, 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) was selected to block the membranes for 1 h at room temperature, which were then incubated with primary antibodies against FLNB (ABclonal Biotechnology Co., Ltd.; 1:500, cat. no. A2481), NLR family apoptosis inhibitory protein (NAIP; BIOSS; 1:1,000; cat. no. bs-5804R), mitogen-activated protein kinase kinase 7 (MAP2K7; ABclonal Biotechnology Co., Ltd.; 1:1,000; cat. no. A2186) and GAPDH (ABclonal Biotechnology Co., Ltd.; 1:50,000; cat. no. AC036) at 4°C overnight with gentle rocking.

Techniques: Expressing, shRNA, Transfection, Plasmid Preparation, MTT Assay, Flow Cytometry, Annexin V Assay, Real-time Polymerase Chain Reaction

Journal: Oncology Reports

Article Title: Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells

doi: 10.3892/or.2020.7532

Figure Lengend Snippet: Validation of FLNB -regulated genes (DEGs). (A) Relative expression level (FPKM, up) and RT-qPCR measurement (down) of cartilage development-related DEGs. (B) Related expression level (FPKM, up) and RT-qPCR measurement (down) of apoptotic-related DEGs. (C) Western blot analysis of two apoptotic-related proteins in shFLNB and Ctrl HeLa cells. FLNB , filamin B; DEGs, differentially expressed genes; FPKM, fragments per kilobase of transcript per million fragments mapped; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin; Ctrl, control; ATP7A, ATPase copper transporting α; BMP7, bone morphogenetic protein 7; COL2A1, collagen type II α 1 chain; MMP13, matrix metallopeptidase 13; IL23A, interleukin 23 subunit α; MALAT1, metastasis associated lung adenocarcinoma transcript 1; NAIP, NLR family apoptosis inhibitory protein; SLC25A36, solute carrier family 25 member 36; MAP2K7, mitogen-activated protein kinase kinase 7.

Article Snippet: Subsequently, 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) was selected to block the membranes for 1 h at room temperature, which were then incubated with primary antibodies against FLNB (ABclonal Biotechnology Co., Ltd.; 1:500, cat. no. A2481), NLR family apoptosis inhibitory protein (NAIP; BIOSS; 1:1,000; cat. no. bs-5804R), mitogen-activated protein kinase kinase 7 (MAP2K7; ABclonal Biotechnology Co., Ltd.; 1:1,000; cat. no. A2186) and GAPDH (ABclonal Biotechnology Co., Ltd.; 1:50,000; cat. no. AC036) at 4°C overnight with gentle rocking.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Molecular Biosciences

Article Title: Drug affinity-responsive target stability unveils filamins as biological targets for artemetin, an anti-cancer flavonoid

doi: 10.3389/fmolb.2022.964295

Figure Lengend Snippet: lmmunoblotting analysis of one of the DARTS experiments revealing FLNA [panel (A)] and FLNB [panel (C) ], together with their densitometric analysis [panels (B,D) , respectively]. GAPDH is resistant to subtilisin under these experimental conditions and is used as a loading control.

Article Snippet: First, 20 µg of each sample were loaded on an 8% SDS-PAGE and transferred onto a nitrocellulose membrane; then, they were incubated for 1 h in a blocking solution (5% w/v milk in TBS-t: 31 mM Tris pH 8, 170 mM NaCl, 3.35 mM KCl, and 0.05% Tween 20) and left for 16 h at 4°C with monoclonal antibodies against FLNA and FLNB (1:1000 v/v, RayBiotech).

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Drug affinity-responsive target stability unveils filamins as biological targets for artemetin, an anti-cancer flavonoid

doi: 10.3389/fmolb.2022.964295

Figure Lengend Snippet: t-LiP-MRM experimental results. ART-protected peptides are reported as blue bars in both FLNA [panel (A)] and FLNB [panel (B)] schematic representations.

Article Snippet: First, 20 µg of each sample were loaded on an 8% SDS-PAGE and transferred onto a nitrocellulose membrane; then, they were incubated for 1 h in a blocking solution (5% w/v milk in TBS-t: 31 mM Tris pH 8, 170 mM NaCl, 3.35 mM KCl, and 0.05% Tween 20) and left for 16 h at 4°C with monoclonal antibodies against FLNA and FLNB (1:1000 v/v, RayBiotech).

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Drug affinity-responsive target stability unveils filamins as biological targets for artemetin, an anti-cancer flavonoid

doi: 10.3389/fmolb.2022.964295

Figure Lengend Snippet: lmmunofluorescence analysis of ART and 8-p-ART-treated (10 and 25 μM; from 24 to 72 h) HeLa cells. These ones were fixed and labeled with an antibody against FLNA (red) and FLNB (green). White arrows show FLNA and FLNB disorganization. Magnification: ×63 1.4 NA. Bar = 100 µm. All images are representative fields of n = 3 experiments with similar results.

Article Snippet: First, 20 µg of each sample were loaded on an 8% SDS-PAGE and transferred onto a nitrocellulose membrane; then, they were incubated for 1 h in a blocking solution (5% w/v milk in TBS-t: 31 mM Tris pH 8, 170 mM NaCl, 3.35 mM KCl, and 0.05% Tween 20) and left for 16 h at 4°C with monoclonal antibodies against FLNA and FLNB (1:1000 v/v, RayBiotech).

Techniques: Labeling

Journal: Oncogenesis

Article Title: Targeting filamin B induces tumor growth and metastasis via enhanced activity of matrix metalloproteinase-9 and secretion of VEGF-A

doi: 10.1038/oncsis.2014.33

Figure Lengend Snippet: H-RAS-transformed Flnb -deficient fibroblasts form larger tumors in SCID mice. ( a ) Genotyping of wild-type ( Flnb +/+ ) and Flnb -deficient ( Flnb −/− ) MEFs transfected with plasmid encoding H-RAS. ( b ) Immunoblots of total RAS expression in Flnb +/+ and Flnb −/− cells transfected with plasmid encoding H-RAS (H-RAS + ) and nontransfected cells (H-RAS − ). ( c ) Number of Flnb −/− H-RAS + , Flnb +/+ H-RAS + and Flnb +/+ H-RAS − (wild-type) MEF cells assayed for proliferation. ( d ) Volume of Flnb −/− H-RAS + and Flnb +/+ H-RAS + tumors inoculated into SCID mice up to 13 days ( n =12 in each group). Data are expressed as mean±s.d. * P <0.05; *** P <0.001 versus Flnb +/+ H-RAS + cells or tumors.

Article Snippet: M2, A7 and OVSCR-8 cells were transfected with either shRNA GFP or FLNB constructs (SA Biosciences, Frederick, MD, USA) using Fugene HD transfection reagent according to the manufacturer's protocol (Promega, Madison, WI, USA).

Techniques: Transformation Assay, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Oncogenesis

Article Title: Targeting filamin B induces tumor growth and metastasis via enhanced activity of matrix metalloproteinase-9 and secretion of VEGF-A

doi: 10.1038/oncsis.2014.33

Figure Lengend Snippet: Increased MMP9 mRNA expression in vascular endothelial and tumor cells in the absence of FLNB. ( a ) Mmp9 mRNA expression in mouse embryonic filamin B-deficient ( Flnb −/− ) ECs as compared with wild-type ( Flnb +/+ ) ECs as detected by reverse transcriptase–PCR (RT–PCR). ( b ) FLNB protein expression in human melanoma M2 cells as detected by immunoblots after transfection with vector expressing FLNB shRNA. Immunoblot with Actin served as loading controls. Transfection with GFP shRNA was included as a negative control. ( c ) Increased mRNA expression of MMP9 in FLNB -silenced M2 cells. ( d ) FLNB protein expression in OVSCR8 cells transfected with FLNB shRNA, whereas same cells transfected with GFP shRNA served as negative controls. ( e ) Proteolytic activity of MMP-9 in human ovarian cancer OVSCR8 cells following transfection with FLNB or GFP shRNA. ( f ) Invasion of OVSCR8 cells after transfection with FLNB or GFP shRNA. Data of triplicated experiments are expressed as mean±s.d. * P <0.05; *** P <0.001 versu s respective controls.

Article Snippet: M2, A7 and OVSCR-8 cells were transfected with either shRNA GFP or FLNB constructs (SA Biosciences, Frederick, MD, USA) using Fugene HD transfection reagent according to the manufacturer's protocol (Promega, Madison, WI, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, shRNA, Negative Control, Activity Assay

Journal: Oncogenesis

Article Title: Targeting filamin B induces tumor growth and metastasis via enhanced activity of matrix metalloproteinase-9 and secretion of VEGF-A

doi: 10.1038/oncsis.2014.33

Figure Lengend Snippet: Increased intratumor vascularity because of increased VEGF-A secretion in Flnb -deficient tumors in vivo . ( a ) Micrographs of vascular networks within mouse tumors formed by H-RAS-transformed MEFs. Red color represents PECAM-positive endothelial cells (scale bar, 100 μm). ( b ) Secreted levels of VEGF-A in mouse H-RAS-transformed wild-type ( Flnb +/+ H-RAS + ) or homozygous Flnb -deficient ( Flnb −/− H-RAS + ) tumors. ( c ) Secreted levels of VEGF-A in wild-type ( Flnb +/+ ) and homozygous Flnb -deficient ( Flnb −/− ) MEFs treated without reagent (Ø), PMA or MAPK inhibitor. ( d ) Secreted levels of VEGF in OVSCR8 cells following transfection with FLNB or GFP shRNA. ( e ) Representative image and quantification of circular vascular structures of porcine aortic endothelial (PAE) cells after conditioning with conditioned medium from FLNB or GFP shRNA-transfected OVSCR8 cells. Data of triplicate or quadruplicate experiments are expressed as mean±s.d. * P <0.05, ** P <0.01 and *** P <0.001 versus respective controls.

Article Snippet: M2, A7 and OVSCR-8 cells were transfected with either shRNA GFP or FLNB constructs (SA Biosciences, Frederick, MD, USA) using Fugene HD transfection reagent according to the manufacturer's protocol (Promega, Madison, WI, USA).

Techniques: In Vivo, Transformation Assay, Transfection, shRNA