Journal: Technology in cancer research & treatment

Article Title: SNU-333 Cells as an Appropriate Cell Line for the Orthotopic Renal Cell Carcinoma Model.

doi: 10.1177/15330338211038487

Figure Lengend Snippet: Figure 4. Quantitative results of cancer stem cell and tumor markers. (A) Representative images of Western blots and densitometry of cancer stem cell and tumor markers. Caki-2 cells showed significantly increased levels of Sox2, c-Myc, and CD105, while Caki-1 cells showed decreased levels of Sox2 compared to HK-2 cells. SNU-333 cells showed significantly increased levels of Sox2, c-Myc, and Lgr5. (B) Representative images of Western blots and densitometry of surface tumor markers. Caki-2 cells showed significantly increased levels of CD24 and CD44, while others showed reduced or comparable levels to HK-2 cells. *P < .05, **P < .01, ***P < .001 versus HK-2 cells.

Article Snippet: The antibodies specific for β-actin (sc-47778; diluted 1:5000), c-Myc (sc-40; diluted 1:1000), E-cadherin (sc-7870; diluted 1:1000), EpCAM (sc-25308; diluted 1:1000), ferritin heavy chain (FTH1; sc-376594; diluted 1:2000), Oct3/4 (sc-5279; diluted 1:1000), SNAIL (sc-271977; diluted 1:1000), Sox2 (sc-365823; diluted 1:1000), TfRC (sc-65882; diluted 1:2000), and vimentin (sc-6260; diluted 1:2000) were purchased from Santa Cruz Biotechnology; CD24 (ab64064; diluted 1:1000), CD44 (ab157107; diluted 1:1000), CD105, EGFR, GPX4, Lgr5, and SLC40A1 (ferroportin, FPN) were purchased from Abcam; and α-smooth muscle actin (A5228; diluted 1:2000; Sigma-Aldrich), CD10 antibody (ready for use; Ventana), CD44-variant 9 (CD44v9/1459; NBP2-53204; diluted 1:1000) (Novus Biologicals), fibronectin (CL54951AP; diluted 1:2000; Cedarlane), PAX8 (ready for use; Roche Diagnostics), and SLC7A11 (cysteine/glutamate transporter [xCT]; ANT-111; diluted 1:1000; Alomone Labs) were purchased as indicated.

Techniques: Western Blot

Journal: International journal of radiation oncology, biology, physics

Article Title: The Olfactory Bulb Provides a Radioresistant Niche for Glioblastoma Cells.

doi: 10.1016/j.ijrobp.2020.01.007

Figure Lengend Snippet: Fig. 3. gH2AX foci in NSC11 xenografts and in vitro radiosensitivity of NSC11 glioblastoma stem-like cells isolated from olfactory bulb and right hemisphere. On day 35 postimplant of NSC11 cells, mice received 6 Gy and were collected for analysis at the indicated time points. (A) Representative images of gH2AX foci in tumor cells (SOX2þ) (40 magnification). The last column corresponds to the overlay used for automated foci counting. (B) gH2AX foci in the corpus callosum, striatum, and olfactory bulb (OB) as a function of time after 6 Gy. Each column corresponds to an individual mouse, with at least 50 cells analyzed per mouse at each location. Bars represent mean standard error of the mean. (C). Two NSC11 tumor-bearing mice were euthanized on day 35 postimplant. Green florescent protein fluorescent tissue was collected from the OB and right hemisphere from each mouse, disaggregated, and seeded into standard tissue culture plates in stem cell media. The resulting neurospheres grown from each location were subjected to in vitro clonogenic survival analysis. Values represent the mean standard deviation of 3 independent experiments.

Article Snippet: The antibodies used were human SOX2 (Cell Signaling), CldU (clone BU1/75, ICR1), phospho-H2AX (Millipore), anti-rabbit-AlexaFluor488, anti-rat-AlexaFluor647, and anti-mouse-AlexaFluor555.

Techniques: In Vitro, Isolation, Standard Deviation

Journal: Current issues in molecular biology

Article Title: Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter.

doi: 10.3390/cimb43020041

Figure Lengend Snippet: Figure 3. Experimental manipulation of Sox2 and Oct4 in SupM2 SORE6 clones. (A) FACS analysis showing the effect on SORE6 reporter activity of overexpressing Sox2 in single-cell clone 5-4 (SupM2 SORE6−). (B) FACS analysis showing the effect on SORE6 reporter activity of overexpressing shSox2 in single-cell clone 6-5 (SupM2 SORE6+). (C) FACS analysis showing the effect of Oct4 downregulation on SORE6 reporter activity in clone 5-4 (SupM2 SORE6−). (D) FACS analysis showing the effect of shOct4 transfection on SORE6 reporter activity in single-cell clone 6-5 (SupM2 SORE6+). All results shown are representative of three independent experiments and were also repeated in a second batch of single-cell clones. Side panels show the fold change of GFP in log scale (mean ± SEM). Bottom panels of each figure are the Western blots showing the effect of transient transfection with Sox2, Oct4, shSox2, and shOct4 plasmids. Empty vector (EV) was included as a negative control. ** p < 0.01, *** p < 0.001.

Article Snippet: The Oct4 expression vector (pLVEF1a-hOCT4-IRES-Neo) was purchased from BiOSETTIA (San Diego, CA, USA). shRNA for Sox2 (#26353) and the Sox2 expression vector (#16577) were purchased from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Activity Assay, Transfection, Western Blot, Plasmid Preparation, Negative Control

Journal: Current issues in molecular biology

Article Title: Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter.

doi: 10.3390/cimb43020041

Figure Lengend Snippet: Figure 6. SORE6−and SORE6+ clones are biochemically distinct. (A) The subcellular localization of Sox2, Oct4, and c-Myc in SORE6−and SORE6+ cells derived from SupM2, assessed by the nuclear cytoplasmic fractionation assay. (B) The DNA pull-down assay was performed to assess Sox2, Oct4, and c-Myc transcriptional activity in SORE6−and SORE6+ cells using a biotin-labeled SORE6 probe.

Article Snippet: The Oct4 expression vector (pLVEF1a-hOCT4-IRES-Neo) was purchased from BiOSETTIA (San Diego, CA, USA). shRNA for Sox2 (#26353) and the Sox2 expression vector (#16577) were purchased from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Derivative Assay, Fractionation, Pull Down Assay, Activity Assay, Labeling

Journal: Journal of cell science

Article Title: Tbl1 promotes Wnt-β-catenin signaling-induced degradation of the Tcf7l1 protein in mouse embryonic stem cells.

doi: 10.1242/jcs.261241

Figure Lengend Snippet: Fig. 1. Overexpression of Tbl genes decreases Tcf7l1 protein levels and is beneficial for maintaining the stemness of mESCs. (A) Western blot analysis of Tcf7l1 protein levels in cells overexpressing Flag tag alone (empty vector control, Flag) or Flag–Tbl1. (B) Densitometric analysis of the relative protein levels of Tcf7l1, as shown in A, was performed with ImageJ software. Protein levels were normalized to those of β-actin. (C) Western blot analysis of Tcf7l1 levels in cells overexpressing Flag tag alone (Flag) or Flag–Tblr1. (D) Densitometric analysis of the relative protein level of Tcf7l1, as shown in C, was performed with ImageJ software. Protein levels were normalized to those of β-actin. (E) RT–qPCR analysis of Tcf7l1 levels in cells overexpressing Flag tag alone (Flag) or Flag–Tbl1. (F) RT–qPCR analysis of Tcf7l1 levels in cells overexpressing Flag tag alone (Flag) or Flag–Tblr1. (G) AP staining of mESCs expressing Flag tag alone, Flag–Tbl1 or Flag–Tblr1 cultured in serum-containing medium without LIF for 8 days. Scale bars: 100 μm. (H) Quantification of AP-positive colonies as shown in G. (I) RT–qPCR analysis of the expression of Oct4, Sox2, Klf4, Esrrb, Nanog and Gata4 in mESCs expressing Flag tag alone, Flag–Tbl1 or Flag–Tblr1. (J) Left: AP staining of mESCs expressing either Flag tag alone or Flag–Tbl1 and transfected with a plasmid encoding HA– Tcf7l1. Cells were cultured in medium containing LIF and serum. Right: immunofluorescence (IF) of HA–Tcf7l1 mESCs transfected with Flag or Flag–Tbl1 showing Oct4 (red) and Hoechst 33342 (blue). IF images are representative of three experiments. Scale bars: 100 μm. (K) Quantification of AP-positive colonies as shown in J. All quantitative data are presented as mean±s.d. of n=3 biological replicates. *P<0.05, **P<0.01 versus Flag, as determined by unpaired, two-tailed Student’s t-test (B,D–F,K) or one-way ANOVA with Sidak’s multiple comparisons test (H,I).

Article Snippet: The primary antibodies used were specific for Flag (F1804, Sigma-Aldrich; 1:2000), Tcf7l1 (bs-12891, Bioss; 1:1000), HA (3724, Cell Signaling Technology; 1:1000), β-actin (66009-1-Ig, Proteintech; 1:1000), β-tubulin (66240-1-Ig, Proteintech; 1:1000), Oct4 (SC-5279, Santa Cruz; 1:1000), Sox2 (66411-1-Ig, Proteintech; 1:1000), Klf4 (381633, ZENBIO; 1:1000), Tbl1 (823294, ZENBIO; 1:1000), Tblr1 (R383062, ZENBIO; 1:1000), non-phospho (Active) β-catenin (Ser33/37/Thr41) (8814, Cell Signaling Technology; 1:1000), β-catenin (SC-7199, Santa Cruz; 1:1000) and ubiquitin (SC-8017, Santa Cruz; 1:1000).

Techniques: Over Expression, Western Blot, FLAG-tag, Plasmid Preparation, Control, Software, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Transfection, Immunofluorescence, Two Tailed Test

Journal: Cell reports

Article Title: Nuclear and cytosolic fractions of SOX2 synergize as transcriptional and translational co-regulators of cell fate.

doi: 10.1016/j.celrep.2024.114807

Figure Lengend Snippet: Figure 3. The SOX2 C-terminal domain modulates protein synthesis in the cytosol (A) Schematic of SOX2 domain separation with indication of DNA-binding high-mobility group (HMG), RNA binding motif (RBM), and transactivation domain (TAD). The indicated demarcation line (red) was chosen to subdivide the human SOX2 protein (residues 1–317, full length) into stably expressing N-terminal (residues 1–179) and C-terminal (residues 180–317) fragments. For extended comparison, a nuclear export sequence (NES) was selectively fused to the C-terminal fragment. (B) Subcellular localization of the indicated SOX2 expression constructs as investigated by life microscopy: mCherry-fusion protein (red), nuclei (Hoechst, blue), and transmitted light (VIS). Scale bar: 10 mm.

Article Snippet: For retrovirus production, 2 mg of pMX vectors carrying Oct4 (Addgene #13366), Klf4 (Addgene #13370), cMyc (Addgene #13375), Sox2 (Addgene #13367) or Sox2 fragment forms thereof and 2 mg of retroviral packaging vector (pCL-Eco, Addgene #12371) were co-transfected into semi-confluent HEK 293T cells (seeded in 10 cm2 dishes 24 h before) using the FuGENE 6 transfection reagent (E2692, Promega) according to the manufacturer’s instructions.

Techniques: Binding Assay, RNA Binding Assay, Stable Transfection, Expressing, Comparison, Sequencing, Construct, Microscopy

Journal: Cell reports

Article Title: Nuclear and cytosolic fractions of SOX2 synergize as transcriptional and translational co-regulators of cell fate.

doi: 10.1016/j.celrep.2024.114807

Figure Lengend Snippet: Figure 5. Functional in vitro validation of SOX2 C terminus-imposed translational adaptations (A) Relative changes in glucose uptake in response to SOX2 (fragment) induction in T47D cells. Indicated are MFI shifts at l = 488 nm (D mean 488 nm) reflecting SOX2 (fragment)-induced changes to the incorporation rate of glucose uptake reporter 2-NBDG. The endogenous uptake rate into DOX-exposed but empty control cells is set to baseline. Dots indicate mean intensity shift in 4 independent experiments. (B) Representative example of a metabolic Seahorse analysis indicating aggravated sugar consumption (followed as extracellular acidification rate [ECAR]) in starved T47D cells expressing either full-length SOX2 (1–317, red) or an NES/C-terminal segment thereof (180–317, green) vs. equally treated empty control cells (black) or T47D cells expressing the SOX2 N terminus (1–179, blue). Dots reflect means of 4 or more technical replicates.

Article Snippet: For retrovirus production, 2 mg of pMX vectors carrying Oct4 (Addgene #13366), Klf4 (Addgene #13370), cMyc (Addgene #13375), Sox2 (Addgene #13367) or Sox2 fragment forms thereof and 2 mg of retroviral packaging vector (pCL-Eco, Addgene #12371) were co-transfected into semi-confluent HEK 293T cells (seeded in 10 cm2 dishes 24 h before) using the FuGENE 6 transfection reagent (E2692, Promega) according to the manufacturer’s instructions.

Techniques: Functional Assay, In Vitro, Biomarker Discovery, Control, Expressing

Journal: Cell reports

Article Title: Nuclear and cytosolic fractions of SOX2 synergize as transcriptional and translational co-regulators of cell fate.

doi: 10.1016/j.celrep.2024.114807

Figure Lengend Snippet: Figure 6. A disease-linked human mutant abolishes the translational role of SOX2 (A) Western blot analyses verify inducibility of the indicated SOX2 variants expressed as mCherry-fusions in T47D. The nucleocytoplasmic transport mutants T116A and T118A, DNA non-binding M49G, and disease-associated SOX2 variants L97P, G130A, and A191T were investigated. Anti-mCherry staining (top) confirms robust induction relative to wild-type transduced cells (blue). Anti-pRPS6(235/236) staining (center) indicates SOX2-imposed changes for all constructs but SOX2(L97P) (red). Anti-RPS6 staining (control, bottom).

Article Snippet: For retrovirus production, 2 mg of pMX vectors carrying Oct4 (Addgene #13366), Klf4 (Addgene #13370), cMyc (Addgene #13375), Sox2 (Addgene #13367) or Sox2 fragment forms thereof and 2 mg of retroviral packaging vector (pCL-Eco, Addgene #12371) were co-transfected into semi-confluent HEK 293T cells (seeded in 10 cm2 dishes 24 h before) using the FuGENE 6 transfection reagent (E2692, Promega) according to the manufacturer’s instructions.

Techniques: Mutagenesis, Western Blot, Binding Assay, Staining, Construct, Control

Journal: Cell reports

Article Title: Nuclear and cytosolic fractions of SOX2 synergize as transcriptional and translational co-regulators of cell fate.

doi: 10.1016/j.celrep.2024.114807

Figure Lengend Snippet: Figure 7. An advanced model of SOX2 in cell fate control (A) SOX2 establishes contact with DNA recognition motifs via the HMG (blue sphere),14 activates these elements by its pioneering TF activity,72 and thus enforces the transcription of cognate mRNAs. (B) To facilitate nuclear export, physical contact with nucleoporin Nup153 may be established and distinct target genes positioned to the nuclear pore.24

Article Snippet: For retrovirus production, 2 mg of pMX vectors carrying Oct4 (Addgene #13366), Klf4 (Addgene #13370), cMyc (Addgene #13375), Sox2 (Addgene #13367) or Sox2 fragment forms thereof and 2 mg of retroviral packaging vector (pCL-Eco, Addgene #12371) were co-transfected into semi-confluent HEK 293T cells (seeded in 10 cm2 dishes 24 h before) using the FuGENE 6 transfection reagent (E2692, Promega) according to the manufacturer’s instructions.

Techniques: Control, Activity Assay