Journal: iScience

Article Title: Cancer cell intrinsic TIM-3 induces glioblastoma progression

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet: TIM-3 is one of the most highly elevated immune checkpoints in GBM and is expressed by both non-tumor and tumor cells (A) The expression analyses of immune checkpoints in GBM, in comparison with non-tumor tissue and LGG, identified 7 overlapping elevated immune checkpoints, including 2 co-inhibitory immune checkpoints ( TIM- 3 and LAIR1 ) and 5 co-stimulatory immune checkpoints ( SLAMF8 , CD300A , TYPOBP , CD58 , and BTN3A2 ) (TCGA-seq, GBM, n = 155; LGG, n = 515; non-tumor, n = 4; GSE16011 , GBM, n = 155; LGG, n = 116; non-tumor, n = 8). (B) RT-qPCR analyses of CTLA4 , TIM3 , LAIR1 , PD1 , PDL1 , PDL2 and ID O 1 in clinical GBM samples (n = 10, means ± SEM, one-way ANOVA). (C) Immunohistochemical staining (Left, scale bar, 50 μm) and analyses (Right, non-tumor, n = 13; Grade II, n = 6; Grade III, n = 17; Grade IV, n = 77; means ± SEM, one-way ANOVA) of TIM-3 in clinical samples. (D) The Kaplan-Meier analyses of clinical glioma samples reveal the correlation of TIM-3 with poor prognosis in GBM (TIM-3 high vs. low, p = 0.0003; log rank test). (E) CIBERSORT analysis of non-tumor cell populations associated with high TIM-3 expression in TCGA RNA-seq GBM dataset (Pearson correlation analysis). (F) The tSNE plot of TIM-3 expression profile in GBM and immune cells with single-cell RNA-seq data ( GSE131928 ). (G) Representative immunofluorescence images of TIM-3 and GFAP staining in clinical GBM samples (scale bar, 50 μm). (H) FACS analysis shows that clinical GBM samples contained low CD45, and high TIM-3 cell populations (n = 7). (I) qPCR analysis of TIM-3 mRNA expression in NHA, indicated glioma cell lines (U87, U251, and LN229), primary glioma cells (PGC1228, PGC21, PGC24, and PGC40), and PBMC from patients with glioma (G-PBMC1 and G-PBMC2) and healthy donor (PBMC1 and PBMC2) (n = 3, means ± SEM, one-way ANOVA). (J) Western blot analyses of TIM-3 in NHA, indicated glioma cell lines, and primary glioma adherent and neurosphere cells (n = 3, means ± SEM, one-way ANOVA). (K) The schematic diagram describing the co-culture system of THP-1-derived anti-inflammatory/pro-tumorigenic (M2) macrophages and glioma cells (left panel), and representative western blot images of TIM-3 in indicated glioma cells cocultured with THP-1-derived anti-inflammatory/pro-tumorigenic TAMs (right panel). (ns p ≥ 0.05, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001).

Article Snippet: Human: U87 cells , GeneChem , RRID: CVCL_0022.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Immunohistochemical staining, Staining, RNA Sequencing, Immunofluorescence, Western Blot, Co-Culture Assay, Derivative Assay

Journal: iScience

Article Title: Cancer cell intrinsic TIM-3 induces glioblastoma progression

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet: TIM-3 increases IL6 expression via activating NF-κB signaling in glioma cells (A) Representative western blotting images of Gal-9 in conditioned medium (CM) from indicated glioma cells separately transfected with TIM-3 overexpression, control, knockdown, or siNC vector. (B) The detection (left) and quantification (right) of cytokines in culture supernatants from GSC40 transduced with TIM-3 overexpression or control vector by proteome profiler cytokine array. (C-E) Representative western blotting images of IL6 in U87 cells transduced with control (U87-NC) or TIM-3 overexpression vectors (U87-TIM3 OE), respectively, and then with indicated treatment at indicated incubation time (C, with CHX; D, with CHX after MG132 pretreatment; E, with CHX after CQ pretreatment). (F) Representative western blot images of indicated glioma cells transduced with TIM-3 overexpression vector with or without blockade of Gal-9 (10 μg/mL). (G) IL6 concentration in CM from indicated cells measured by ELISA (n = 3, means ± SEM, one-way ANOVA). (H) Representative western blot images of indicated cells incubated with indicated concentrations (ng/mL) of IL6. (I-K) The decreased growth (I, n = 3, means ± SEM, one-way ANOVA; scale bar, 50 μm), migration (J, n = 3, means ± SEM, one-way ANOVA; scale bar, 50 μm), and neurosphere formation abilities (K, upper: n = 10, extreme limiting dilution assay; lower: stem cell frequency, n = 3, means ± SEM, one-way ANOVA) of GSC40 induced by TIM-3 knockdown was partially rescued by IL6 supplement (40 ng/mL). (ns p ≥ 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: Human: U87 cells , GeneChem , RRID: CVCL_0022.

Techniques: Expressing, Western Blot, Transfection, Over Expression, Control, Knockdown, Plasmid Preparation, Transduction, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Migration, Limiting Dilution Assay

Journal: iScience

Article Title: Cancer cell intrinsic TIM-3 induces glioblastoma progression

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet:

Article Snippet: Human: U87 cells , GeneChem , RRID: CVCL_0022.

Techniques: Control, Purification, Recombinant, Modification, Sterility, Cell Culture, Lysis, Transfection, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Imaging, Sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software, Fluorescence, Microscopy

Journal: Amino Acids

Article Title: Arginine deprivation affects glioblastoma cell adhesion, invasiveness and actin cytoskeleton organization by impairment of β-actin arginylation

doi: 10.1007/s00726-014-1857-1

Figure Lengend Snippet: Effect of arginine deprivation on glioblastoma cells. a Cell growth of U251 ( upper panel ) and U87 ( lower panel ) cells cultivated in control, -Arg, -Lys and conditions. b U251 cell viability assessed under deprivation and re-supplementation conditions, as indicated. Upper and lower panels , arginine and lysine re-supplementation, respectively. 100 %, the number of the viable cells at time 0. Data in a and b are means ± SD; *** and *Statistical relevance p < 0.001 and p < 0.05, respectively

Article Snippet: Human glioblastoma U251 MG (U251) and U87 MG (U87) cell lines were purchased from CLS Cell Lines Service (Germany) and monitored for correct genetic profile via microsatellite analyses according to a protocol described earlier (Peickert et al. ).

Techniques:

Journal: Amino Acids

Article Title: Arginine deprivation affects glioblastoma cell adhesion, invasiveness and actin cytoskeleton organization by impairment of β-actin arginylation

doi: 10.1007/s00726-014-1857-1

Figure Lengend Snippet: Arginine deprivation affects morphology of glioblastoma but not glia cells. a , b , d Rat glia, U251 and U87 cells stained with Alexa 488-phalloidin, respectively. c Micrographs of U251 cells attained with scanning electron microscope. Insets in a , b and d ~2–3× magnification of the marked areas . Bars , in a , b and d 50 μm, and in c 10 μm. e U251 cells stained with Alexa 488-phalloidin before and after re-supplementation with Arg or Lys up to 0.4 and 0.8 mM concentration, respectively. Arrows point to lamellipodia, arrowheads point to elongated cells

Article Snippet: Human glioblastoma U251 MG (U251) and U87 MG (U87) cell lines were purchased from CLS Cell Lines Service (Germany) and monitored for correct genetic profile via microsatellite analyses according to a protocol described earlier (Peickert et al. ).

Techniques: Staining, Microscopy, Concentration Assay

Journal: Amino Acids

Article Title: Arginine deprivation affects glioblastoma cell adhesion, invasiveness and actin cytoskeleton organization by impairment of β-actin arginylation

doi: 10.1007/s00726-014-1857-1

Figure Lengend Snippet: Arginine deprivation impairs cell motility. a , b Migration tracks of U251 and U87 cells, respectively. Upper panels in a and b tracks of 10 randomly chosen cells; center panels images of migrating cells, and lower panels values of migration rate and mean distance based on tracks shown in upper panels . Values are means ± SD. ***Statistical relevance p < 0.001

Article Snippet: Human glioblastoma U251 MG (U251) and U87 MG (U87) cell lines were purchased from CLS Cell Lines Service (Germany) and monitored for correct genetic profile via microsatellite analyses according to a protocol described earlier (Peickert et al. ).

Techniques: Migration

Journal: Amino Acids

Article Title: Arginine deprivation affects glioblastoma cell adhesion, invasiveness and actin cytoskeleton organization by impairment of β-actin arginylation

doi: 10.1007/s00726-014-1857-1

Figure Lengend Snippet: Arginine deprivation impairs cell migration and invasiveness. Transwell filters not covered ( a ), and covered with Matrigel ( b , c) were used for analyses. Upper and lower panels in a and b images of U251 and U87 stained cells, respectively, taken on the filter trans side. c Images of LN-229 cells, analyzed as in b . Analyses were performed for three independent experiments run in duplicates. d Images of GFP-expressing U251 cells found within the E13 organotypic brain slice. The images represent the confocal 12.3-μm z -section of the planar center of brain slices. Right panel the quantification of GFP-expressing U251 cells within the confocal center of the slice per view area. The quantitative data in a – d are presented as % of control. Values are means ± SD. ***Statistical relevance p < 0.001

Article Snippet: Human glioblastoma U251 MG (U251) and U87 MG (U87) cell lines were purchased from CLS Cell Lines Service (Germany) and monitored for correct genetic profile via microsatellite analyses according to a protocol described earlier (Peickert et al. ).

Techniques: Migration, Staining, Expressing, Slice Preparation