human u373  (ATCC)

Journal: iScience

Article Title: USP25 deubiquitinates cytosolic METTL3 to impede glioma proliferation via an m6A-independent pathway

doi: 10.1016/j.isci.2025.113918

Figure Lengend Snippet: USP25 deubiquitinates and stabilizes cytoplasmic METTL3 (A–F) The protein levels of METTL3 and USP25 in indicated U87-MG cells and MEF cells were detected by Western blotting (A and D), with m6A levels in poly(A)+ RNA were detected by dot blot analysis, with methylene blue staining as a loading control (B and E), with the quantification of m6A/A ratios in poly(A)+ RNA were measured by LC-MS analysis ( n = 3 biological replicates) (C and F). (G) Western blot analysis of the distribution of METTL3 and USP25 in nuclear and cytoplasmic fractions from U87-MG, U251, A172, and U373 cells. (H) Endogenous USP25 (green) and METTL3 (red) in USP25 +/+ and USP25 −/− mixed MEF cells were detected by immunofluorescence staining. Nuclei were visualized with DAPI (blue). Scale bars, 10 μm. (I) Western blot analysis of METTL3 and USP25 distribution in nuclear and cytoplasmic fractions of U87-MG sgGFP control or sgMETTL3 (#1 and #2) cells. (J) Flag-METTL3 stably expressing U87-MG cells infected with lentivirus expressing sgGFP control or sgUSP25 (#1 and #2) were treated with MG-132 for 6 h. Total lysates, nuclear, and cytoplasmic fractions were immunoprecipitated with Flag antibody, and polyubiquitylated Flag-METTL3 was detected by anti-ubiquitin antibody. Data are represented as mean ± SEM. Statistical analyses using unpaired t test were performed on GraphPad Prism, where ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: Human: U373 , ATCC , HTB-17.

Techniques: Western Blot, Dot Blot, Staining, Control, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Stable Transfection, Expressing, Infection, Immunoprecipitation, Ubiquitin Proteomics

Journal: Biomolecules

Article Title: Cell Settling, Migration, and Stochastic Cancer Gene Expression Suggest Potassium Membrane Flux May Initiate pH Reversal

doi: 10.3390/biom15081177

Figure Lengend Snippet: Migration of glioblastoma cell lines to hepatocyte growth factor (HGF) and fetal bovine serum (FBS). ( A ) Representative Boyden assays of 2 rat astrocytoma (C6 and F98) and 6 glioblastoma cell lines (T98G, LN18, U87, LN229, A172, and U373) showed background (unstimulated) cell migration (row 1), cell migration to HGF (row 2), HGF and FBS (rows 3–9), and to FBS alone (rows 10–12) using concentrations in the bottom wells as indicated on the left. With the same amount of HGF present, adding increasing amounts of FBS increased chemotactic cell migration in all cell lines except A172. Images were obtained with a transparency scanner. ( B ) Portions of assays (regular scanner) with FBS alone as the chemoattractant were digitized for densitometry, with results (average total pixels) shown. All cell lines showed significant migration to FBS except A172, which only had a trend, p = 0.0638, for 0.5% FBS. All assays (3 per cell line) are in , for C6, F98, T98G, and LN18 and U87, LN229, A172, and U373 . ( C ) Immunoblot with densitometry (total pixels) underneath quantifies HGF’s receptor, Met (β subunit, 145 kDa), among cell lines. For comparison, HGF-stimulated cell migration is below the bar graph as times (X) background cell migration and lacks notable correlation. The immunoblot’s gel is in ( A). ( D ) MEM as media in the bottom wells, with and without FBS, outperformed Dulbecco’s PBS for U87 cell migration with “Migration” media in all top wells. Migration was 1.7X, 1.1X, and 1.4X greater (significantly, p < 0.05) using MEM compared to PBS for 0, 0.1, and 1% FBS in the bottom wells, respectively. MEM’s greater potassium content (5.3 mM) in the bottom wells than in “Migration” media (4.5 mM K + ; see text) in the top wells putatively provided a chemoattractant K + gradient. p =0.0144 in MEM versus Dulbecco’s PBS with 0% FBS, and with 0.1 and 1% FBS it was also present, p = 0.0136 and 0.000422, respectively. The scanned image of the assay is in ( B). ( E ) Chemotaxis to HGF and FBS outperformed chemokinesis and FBS enhanced HGF’s effects on U87 cells in MEM (top and bottom wells). Chemotaxis was consistently greater than chemokinesis in paired comparisons designated by brackets under the x-axis with significance, p = 0.012, for row 8 versus row 9. The assay image is in ( C). Averages of rows’ densitometry units indicated by bar heights with 95% confidence intervals. Variable staining of background migration and assay-to-assay variation for each cell line in are routine. Each circle of migrated cells is 3 mm in diameter.

Article Snippet: A172 (RRID:CVCL_0131), C6 (TKG Cat# TKG 0242, RRID:CVCL_0194), F98 (ATCC Cat# CRL-2397, RRID:CVCL_3510), LN18 (RRID:CVCL_0392), LN229 (RRID:CVCL_0393), T98G (RRID:CVCL_0556), U87 (RRID:CVCL_0022), and U373 (RRID:CVCL_2219) glioblastoma/astrocytoma cell lines (human (A172, LN18, LN229, U87, U373) and rat (F98 and C6)) were obtained from the American Type Culture Collection, Manassas, VA, USA, RRID:SCR_021346.

Techniques: Migration, Western Blot, Comparison, Chemotaxis Assay, Staining

Journal: Biomolecules

Article Title: Cell Settling, Migration, and Stochastic Cancer Gene Expression Suggest Potassium Membrane Flux May Initiate pH Reversal

doi: 10.3390/biom15081177

Figure Lengend Snippet: All three migration assays for each cell line (U87, LN229, A172, and U373) in studies of HGF and FBS chemoattraction. ( A ) Three migration assays for U87 and LN229 cell line. ( B ) Three migration assays for A172 and U373 cell line. One assay per cell line is shown in A. Portions of assays that were digitized and analyzed are shown in B. Text in . and the legend provides additional information. Each circle of stained migrated cells is 3 mm in diameter.

Article Snippet: A172 (RRID:CVCL_0131), C6 (TKG Cat# TKG 0242, RRID:CVCL_0194), F98 (ATCC Cat# CRL-2397, RRID:CVCL_3510), LN18 (RRID:CVCL_0392), LN229 (RRID:CVCL_0393), T98G (RRID:CVCL_0556), U87 (RRID:CVCL_0022), and U373 (RRID:CVCL_2219) glioblastoma/astrocytoma cell lines (human (A172, LN18, LN229, U87, U373) and rat (F98 and C6)) were obtained from the American Type Culture Collection, Manassas, VA, USA, RRID:SCR_021346.

Techniques: Migration, Staining

Journal: Biomolecules

Article Title: Cell Settling, Migration, and Stochastic Cancer Gene Expression Suggest Potassium Membrane Flux May Initiate pH Reversal

doi: 10.3390/biom15081177

Figure Lengend Snippet: Many KCN DEGs detected in E1–E70 studies ( , and ) and in REMBRANDT were included in whole-genome and expression studies of 6 glioblastoma cell lines, LN18, LN229, T98G, U87, U343, and U373, by Patil et al. . For comparisons, shared KCN DEGs in E1–E70 (29 cancers including 7 glioblastomas) are marked with green squares and KCN genes of interest found in REMBRANDT are bolded ( A , B ). ( A ) Sequencing revealed DNA alterations including cancer-specific mutations (CSMs), insertions (INSs), and deletions (DELs). A total of 23 KCN DEGs (of 53 in E1–E70; see and ) and those of interest in REMBRANDT (3 with trends and 1 significantly differentially expressed; see C–F) had changes in genomes of 6 cell lines reported by Patil et al. Several shared KCN DEGs were genetically altered in all 6 cell lines. ( B ) Average RNA expressions determined by Patil et al. are listed in descending order of abundance, including 27 and 4 of E1–E70 KCN DEGs and REMBRANDT’s KCN DEGs and genes of interest, respectively, with names of proteins. ( C ) Log2 fold RNA changes in combined tumor cell lines versus normal (see text) are shown with KCNs encoding H + -sensitive proteins indicated with red (H + ) and KCNs with DNA changes in ( A ) with underlying blue brackets (˽). Of 51 KCNs in the glioblastoma cell lines, 27 (52.9%) encoded H + -sensitive proteins. Of 53 KCNs in E1–E70, 38 (71.7%) were found in their glioblastoma cell lines, with altered DNA and/or RNA expression. Of seven GBM studies in E1–E70, six (85.7%) shared 1–3 KCN DEGs with those described by Patil et al. . See the text and ( A) for specific glioblastoma studies in E1–E70. References for studies are in .

Article Snippet: A172 (RRID:CVCL_0131), C6 (TKG Cat# TKG 0242, RRID:CVCL_0194), F98 (ATCC Cat# CRL-2397, RRID:CVCL_3510), LN18 (RRID:CVCL_0392), LN229 (RRID:CVCL_0393), T98G (RRID:CVCL_0556), U87 (RRID:CVCL_0022), and U373 (RRID:CVCL_2219) glioblastoma/astrocytoma cell lines (human (A172, LN18, LN229, U87, U373) and rat (F98 and C6)) were obtained from the American Type Culture Collection, Manassas, VA, USA, RRID:SCR_021346.

Techniques: Expressing, Sequencing, RNA Expression