Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original tumor, low passage, and high passage KCI-MENG1 cells. The original patient-derived tumor ( top row ) showed moderate and patchy immunoreactivity for epithelial membrane antigen (EMA); strong and diffuse immunostaining for progesterone receptor (PR); and a Ki-67 proliferative index of 2–3%. There was also strong immunostaining for N-cadherin and vimentin. KCI-MENG1-LP cells ( middle row ) and KCI-MENG1-HP cells ( bottom row ) maintained expression of EMA, N-cadherin, and vimentin but had significantly reduced PR expression compared to the original tumor. Whereas Ki-67 labeling was found in only a small number of cells in the original tumor and low passage cells, it was positive in virtually all P84 cells. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, Membrane, Expressing, Labeling

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original patient tumor, low and high passage KCI-MENG1 cells, and subcutaneous xenograft tumor. The original patient-derived tumor showed moderate immunoreactivity for E-cadherin which was maintained in all in vitro and in vivo models. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, In Vitro, In Vivo

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Meningioma cell lines reported in the literature

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Southern Blot, Injection, Expressing, TRAP Assay, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Human meningioma mouse xenograft model KCI-MENG1-LPSX generated with the spontaneously immortal cell line KCI-MENG1-LP. Tumors from immunocompromised SCID mice were dissected ( a ) and the derivative cell line KCI-MENG1-LPSX CL was generated. The H&E staining of the mouse tumor revealed a pattern of moderately cellular meningothelial cells similar to the original patient tumor ( b ). The KCI-MENG1-LPSX CL cells were composed of the round-shaped cells similar to the high passage parent cell line KCI-MENG1-HP ( c ). The EMA, PR, and N-cadherin IHC of the mouse tumor highly resembled the original patient-derived tumor ( d top row ). The vimentin- and Ki-67-stained cells in the mouse tumor tissue were markedly more abundant and more intensely stained than in the original tumor ( d top row ). KCI-MENG1-LPSX CL cells displayed the same patterns of immunostaining as the high passage parent cell line KCI-MENG1-HP, including the loss of PR staining ( d bottom row ). Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Generated, Staining, Derivative Assay, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Depletion of Lipocalin 2 (LCN2) in Mice Leads to Dysbiosis and Persistent Colonization with Segmented Filamentous Bacteria

doi: 10.3390/ijms222313156

Figure Lengend Snippet: Comparative analysis of the intestine macroscopical parameters and expression of Lcn2 in wild type (n = 4) and Lcn2 -/- mice (n = 4 or 5). ( A ) Evaluation of the body weight of male animals at the same age (left). Corresponding ceca were prepared and weighed (middle). Ratio of body to cecal weight (right). ( B ) Gross macroscopical and graphical comparison of the total small and large intestinal lengths measured (n = 4 or 5). ( C ) Quantitative analysis of Lcn2 mRNA (upper) and LCN2 protein expression (lower) in the distal small intestine (left) cecum (middle), and distal colon (right) of wild type ( Lcn2 +/+ ) and Lcn2 null mice (each n = 4 or 5). Relative mRNA expression of respective genes was done by RT-qPCR and normalized to Gapdh or β-actin . Equal protein loading was demonstrated by re-probing the membranes with a specific antibody directed against GAPDH or β-actin. E-cadherin was probed as a marker for epithelial-to-mesenchymal transition (EMT). Primers used are given in . Statistical evaluation in ( A – C ) was performed by an unpaired t -test, whereby the difference of two samples from a level of p < 0.05 is considered statistically significant (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

Article Snippet: For detection of individual proteins, the primary antibodies for LCN2/NGAL (AF3508, R & D Systems, Abingdon, UK), GAPDH (6C5) (sc-32233, Santa Cruz, Biotech, Santa Cruz, CA, USA), β-actin (A5441, Sigma–Aldrich, MI, USA), and E-cadherin (AP20195PU-N, OriGene, Rockville, MD, USA) were diluted in 2.5% ( w / v ) non-fat milk powder in TBST.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Marker

Journal: Journal of Biological Chemistry

Article Title: Klotho Inhibits Transforming Growth Factor-β1 (TGF-β1) Signaling and Suppresses Renal Fibrosis and Cancer Metastasis in Mice

doi: 10.1074/jbc.m110.174037

Figure Lengend Snippet: FIGURE 9. Secreted Klotho inhibits EMT in A549 cancer cells. A, Klotho inhibits TGF-1-induced phosphorylation of Smad3 in A549 cells. A549 cells were incubated with secreted Klotho protein for 15 min and then stimulated with TGF-1 for 15 min at the indicated doses. Cell lysates were subjected to immunoblot analyses using antibody against phosphorylated Smad3 (pSmad3) or antibody that recognized Smad3 regardless of its phosphoryla- tion state (Smad3). B, Klotho inhibits TGF-1-induced activation of the Smad- responsive reporter. A549 cells were transfected with a luciferase reporter containing Smad response elements (pGTCT2 2-Luc) and a lacZ expression vector for normalization. These cells were incubated with TGF-1 or Klotho at the indicated doses for 6 h and subjected to a standard luciferase assay. The luciferase activity was normalized with that of non-treated cells. Data indicate means S.E. of three independent experiments. p 0.02 by one-way ANOVA. C, Klotho inhibits TGF-1 binding to A549 cells. TGF-1 binding assays were performed in A549 cells in the absence or presence of Klotho protein (0.1 or 0.3 nM). The amount of bound TGF-1 was normalized with that without Klotho. Data indicate means S.E. of four independent experi- ments. p 0.006 by one-way ANOVA. D, Klotho protein suppresses TGF-1- induced decrease in E-cadherin and increase in N-cadherin. A549 cells were treated with TGF-1 and/or Klotho at the indicated doses for 6 h and then subjected to immunoblot analyses 48 h later. E, Klotho protein suppresses TGF-1-induced cell migration. A549 cells were treated with TGF-1 and/or Klotho at the indicated doses for 6 h and then subjected to a standard Tran- swell migration assay. Data indicate means S.E. of three independent experiments. *, p 0.001 versus TGF-1 treatment alone by two-tailed t test.

Article Snippet: Primary antibodies used in this study were Klotho (KM2119)(30), SMA, Vimentin (Santa Cruz Biotechnology), E-Cadherin (BD Biosciences, San Jose, CA), N-cadherin (Santa Cruz Biotechnology), phosphorylated Smad2 (Cell Signaling Technology, Beverley, CA), phosphorylated Smad3 (Cell Signaling), Smad2 (Cell Signaling), Smad3 (Cell Signaling), GAPDH (Abcam).

Techniques: Phospho-proteomics, Incubation, Western Blot, Activation Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Binding Assay, Migration, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: N-acetyltransferase 10 promotes glioblastoma malignancy via mRNA stabilization of jumonji and AT-rich interaction domain containing 2

doi: 10.1016/j.jbc.2025.108544

Figure Lengend Snippet: Suppression of GBM stemness by knockout of NAT10 . A , the proliferation of naive and NAT10 knockout (KO) U251 and A172 cells. Values show the mean with S.D. (n = 12). The cell viability of seeding day (day 0) was set at 1.0. ∗∗; p < 0.01 significant difference from naive group at corresponding time points. ( F 9,110 = 69.073 p < 0.001 for U251 cells, F 9,110 = 2331.346 p < 0.001 for A172 cells; ANOVA with the Tukey-Kramer post hoc test). Each upper panel shows the NAT10 protein levels in naive and NAT10 KO U251 and A172 cells. B , the spheroid formation ability of naive and NAT10 KO U251 and A172 cells. Each left panel shows a representative photograph of Hoechst33342-stained spheroids-formed by naive or NAT10 KO U251 or A172 cells. Each right panel shows the number of spheroids and the distribution of their diameters. Values show the mean with S.D. (n = 5 for U251 cells, n = 4 for A172 cells). ∗∗; p < 0.01, significant difference between the two groups ( t = 13.355 for U251 cells, t = 2.663 for A172 cells, Welch’s t test). C , the invasion ability of naive and NAT10 KO U251 and A172 cells. Microphotographs show invasion of cells into 3D collagen gel. D , the sensitivity of naive and NAT10 KO U251 and A172 cells to temozolomide. Naive and NAT10 KO cells were treated with indicated concentration of temozolomide for 96 h. Values show the mean with S.D. (n = 8 for U251 cells, n = 6 for A172 cells). Cell viability of vehicle-treated groups was set at 1.0. ∗∗; p < 0.01, ∗; p < 0.05; significant difference between the two groups at corresponding concentration points. ( F 7 ,56 = 55.596 p < 0.001 for U251 cells, F 7,40 = 7.381 p < 0.001 for A172 cells; ANOVA with the Tukey-Kramer post hoc test). E , protein expression levels in SOX2, OCT4, KLF4 in naive or NAT10 KO U251 cells. Protein levels were normalized to those of p84 expression levels. Values show the mean with S.D. (n = 5–6). ∗∗; p < 0.01, ∗; p < 0.05; significant difference between the two groups ( t 10 = 4.134 for SOX2, t 10 = 3.001 for OCT4, t 8 = 4.491 for KLF4, Student’s t test). F , protein expression levels in N-CADHERIN, VIMENTIN in naive and NAT10 KO U251 cells. Protein levels were normalized to those of β-ACTIN expression levels. Values show the mean with S.D. (n = 6). ∗∗; p < 0.01 significant difference between the two groups ( t 10 = 3.979 for N-CADHERIN, t 10 = 5.635 for VIMENTIN, Student’s t test).

Article Snippet: Membranes were reacted with antibodies against NAT10 (13365-1-AP, Proteintech, Wuhan, China, RRID:AB_2148944), p84 (THOC1, 10920-1-AP, Proteintech, RRID:AB_2202239), SOX2 (AF2018, R&D systems, RRID:AB_355110), KLF4 (#4038, Cell Signaling Technology, RRID:AB_2265207), OCT4 (#4038, Cell Signaling Technology), N-CADHERIN (AF6426, R&D systems, RRID:AB_10718850), VIMENTIN (MAB21052, R&D systems, RRID:AB_2832972), SUZ12 (#3737, Cell Signaling Technology, RRID:AB_2196850), JARID2 (#13594, Cell Signaling Technology, RRID:AB_2798269), THRAP3 (TRAP150, sc-133250, Santa Cruz Biotechnology, RRID:AB_2202901), MAD1L1 (MAD1, 18322-1-AP, Proteintech, RRID:AB_2139251) and β-ACTIN conjugated with horseradish peroxidase (sc-47778, Santa Cruz Biotechnology, RRID:AB_2202239).

Techniques: Knock-Out, Staining, Concentration Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: N-acetyltransferase 10 promotes glioblastoma malignancy via mRNA stabilization of jumonji and AT-rich interaction domain containing 2

doi: 10.1016/j.jbc.2025.108544

Figure Lengend Snippet: Contribution of JARID2 to the maintenance of stemness properties of GBM cells . A , decrease in the growth ability of U251 and A172 cells by downregulation of JARID2. Each upper panel show JARID2 protein levels in U251 and A172 cells transfected with shRNA against JARID2 or scramble shRNA (shScramble). Each below graph shows the cell viability of seeding day (day 0) was set at 1.0. Values show the mean with S.D. (n = 6). ∗∗; p < 0.01 significant difference from shScramble group at corresponding time points. ( F 9,50 = 1636.125 p < 0.001 for U251 cells, F 9,50 = 140.977 p < 0.001 for A172 cells, ANOVA with the Tukey-Kramer post hoc test). B , the spheroid formation ability of U251 and A172 cells transfected with shRNA against JARID2 or shScramble. Eash left panel shows a representative photograph of Hoechst33342-stained spheroids formed by shScramble- or shJARID2-transfected U251 and A172 cells. Right panel shows the number of spheroids and the distribution of their diameters. Values show the mean with S.D. (n = 5–6 for U251 cells, n = 8 for A172 cells). ∗∗; p < 0.01 significant difference between the two groups ( t = 11.897. for U251 cells, t = 3.412 for A172 cells, Welch’s t test). C , the invasion ability of U251 and A172 cells transfected with shRNA against JARID2 or shScramble. Microphotographs show invasion of cells into 3D collagen gel. D , the protein expression levels of SOX2, OCT4, and KLF4 in U251 cells transfected with shRNA against JARID2 or scramble shRNA (shScramble). Values of protein levels were normalized to p84 protein levels. Values show the mean with S.D. (n = 5). ∗∗; p < 0.01 significant difference between the two groups ( t 8 = 3.407 for SOX2, t 8 = 12.072 for KLF4, Student’s t test). E , the protein expression levels N-CADHERIN and VIMENTIN in shScramble- or shJARID2-transfected U251 cells. Values of protein levels were normalized to those of β-ACTIN levels. Values show the mean with S.D. (n = 5). ∗∗; p < 0.01 significant difference between the two groups ( t 8 = 9.389 for N-CADHERIN, t 8 = 6.029 for VIMENTIN, Student’s t test). F , decrease in the malignancy of U251 cells by downregulation of JARID2. shScramble or shJARID2 RNA expressing lentivirus transfected U251 cells were subcutaneously implanted in Balb/c-nude mice. Left graph shows Kaplan-Meier survival curves shScramble or shJARID2 RNA expressing lentivirus transfected U251 tumor-bearing mice (n = 7 for shScramble, n = 9 for shJARID2). Right graph shows the tumor volume of each individual mouse. ∗; p < 0.05 significant difference between the two groups (LogRank Holm-Sidak test).

Article Snippet: Membranes were reacted with antibodies against NAT10 (13365-1-AP, Proteintech, Wuhan, China, RRID:AB_2148944), p84 (THOC1, 10920-1-AP, Proteintech, RRID:AB_2202239), SOX2 (AF2018, R&D systems, RRID:AB_355110), KLF4 (#4038, Cell Signaling Technology, RRID:AB_2265207), OCT4 (#4038, Cell Signaling Technology), N-CADHERIN (AF6426, R&D systems, RRID:AB_10718850), VIMENTIN (MAB21052, R&D systems, RRID:AB_2832972), SUZ12 (#3737, Cell Signaling Technology, RRID:AB_2196850), JARID2 (#13594, Cell Signaling Technology, RRID:AB_2798269), THRAP3 (TRAP150, sc-133250, Santa Cruz Biotechnology, RRID:AB_2202901), MAD1L1 (MAD1, 18322-1-AP, Proteintech, RRID:AB_2139251) and β-ACTIN conjugated with horseradish peroxidase (sc-47778, Santa Cruz Biotechnology, RRID:AB_2202239).

Techniques: Transfection, shRNA, Staining, Expressing