Journal: Cell death discovery

Article Title: Stanniocalcin 2 drives malignant transformation of human glioblastoma cells by targeting SNAI2 and Matrix Metalloproteinases.

doi: 10.1038/s41420-022-01090-6

Figure Lengend Snippet: Fig. 1 Aggressive GBM expresses higher level of STC2. a, b Representative tissue microarray data of STC2 in clinical specimens (a, GL2082; b, GL208, Biomax). The scale bar represents 100 µm. STC2 staining score from 0-4 were analyzed in different tissues. N, normal tissues; A, astrocytoma; GBM, glioblastoma. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, n.s., no significance. c The levels of STC2 mRNA expression in low grade glioma (LGG) versus glioblastoma (GBM) tissues obtained from TCGA Research Network were analyzed. Unpaired t- test, **p < 0.01. d The mRNA expression of STC2 in various glioblastoma cell lines were analyzed. After normalizing with 18 S rRNA in each sample, the relative mRNA levels were calculated using A172 = 1. Means ± SD; n = 3 biological replicates. Oneway ANOVA, **p < 0.01, ***p < 0.001, n.s., no significance. e, f Representative images of Western blotting against STC2 and β-ACTIN in whole lysates of glioblastoma cell lines (e) and concentrated culture media (Conditioned media CM) (f).

Article Snippet: Human recombinant STC2 (Cat#9405-SO) was purchased from R&D Systems.

Techniques: Microarray, Staining, Expressing, Western Blot

Journal: Cell death discovery

Article Title: Stanniocalcin 2 drives malignant transformation of human glioblastoma cells by targeting SNAI2 and Matrix Metalloproteinases.

doi: 10.1038/s41420-022-01090-6

Figure Lengend Snippet: Fig. 2 Overexpression of STC2 results in invasive phenotypes of GBM cell lines. a, b STC2 mRNA (a) and protein (b) expression was validated after modulation of STC2. LN18 cell was transfected with pRS-shSTC2 to knockdown STC2 whereas A172 cell was transfected with Myc-DDK- tagged-STC2 to overexpress STC2. Means ± SD; n = 3 biological replicates; Student’s two tailed t-test, ***p < 0.001. c Cells were seeded to 6-well plates at a density of 1000 cells per well and colony formation was determined after 10 days. Absorbance at 590 nm was measured after dissolving with 10% acetic acid. Means ± SD; n = 3 biological replicates; Student’s two tailed t-test, ***p < 0.001. d Cell growth rates after STC2 modulation were determined by MTT assay. Means ± SD; n = 12 biological replicates. Student’s two tailed t-test, *p < 0.05, ***p < 0.001. e In vitro invasion and motility were determined onto Matrigel-coated or non-coated Transwell chambers for 48 h. Scale bar = 100 µm. Means ± SD; n = 5 biological replicates; Student’s two tailed t-test, *p < 0.05, ***p < 0.001. f In vitro migration was determined for 24 h after STC2 modulation. Scale bar = 100 µm.

Article Snippet: Human recombinant STC2 (Cat#9405-SO) was purchased from R&D Systems.

Techniques: Over Expression, Expressing, Transfection, Knockdown, Two Tailed Test, MTT Assay, In Vitro, Migration

Journal: Cell death discovery

Article Title: Stanniocalcin 2 drives malignant transformation of human glioblastoma cells by targeting SNAI2 and Matrix Metalloproteinases.

doi: 10.1038/s41420-022-01090-6

Figure Lengend Snippet: Fig. 3 Secreted STC2 induces invasive phenotypes of neighboring GBM cells. a Cell growth rates after treatment of recombinant STC2 (100 ng/mL) or STC2-containing CM were determined using MTT assay. Means ± SD; n = 12 biological replicates. Student’s two tailed t-test, *p < 0.05, **p < 0.01, ***p < 0.001. Significance was analyzed against shSTC2 in LN18, Con in A172. b, c) Colony formation (b) and in vitro invasion (c) were determined after treatment of recombinant STC2 (100 ng/mL) or STC2-containing CM. Absorbance at 590 nm was measured after dissolving with 10% acetic acid. Means ± SD; n = 3 biological replicates; One-way ANOVA, **p < 0.01, ***p < 0.001. d In vitro migration was determined after treatment of recombinant STC2 (100 ng/mL) or STC2-containing CM. Scale bar = 100 µm. e Migrating cells were visualized using fluorescent phalloidin (green) and DAPI (blue) staining. Scale bar = 50 µm.

Article Snippet: Human recombinant STC2 (Cat#9405-SO) was purchased from R&D Systems.

Techniques: Recombinant, MTT Assay, Two Tailed Test, In Vitro, Migration, Staining

Journal: Cell death discovery

Article Title: Stanniocalcin 2 drives malignant transformation of human glioblastoma cells by targeting SNAI2 and Matrix Metalloproteinases.

doi: 10.1038/s41420-022-01090-6

Figure Lengend Snippet: Fig. 4 STC2 targets SNAI2 and MMPs. a SNAI2, MMP-2, and MMP-9 mRNA expressions were validated after modulation of STC2 in LN18 or A172 cell lines. Means ± SD; n = 3 biological replicates; Student’s two tailed t-test, *p < 0.05, **p < 0.01, ***p < 0.001. b SNAI2 protein expression was validated after modulation of STC2. c Proteolytic activities of MMP-2 and MMP-9 were determined by gelatin zymography after STC2 modulation. d The correlation of SNAI2 expression with STC2 (left), the correlation of MMP-2 expression with SNAI2 (middle), and the correlation of MMP-9 expression with SNAI2 (right) were determined in the TCGA data. e SNAI2, MMP-2, and MMP-9 mRNA expressions were measured after treatment of recombinant STC2 (50, 100 ng/mL) or STC2-containing CM (50%). Means ± SD; n = 3; One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, n.s., no significance. f SNAI2 protein expression was validated after treatment of recombinant STC2 (50, 100, 200 ng/mL) or STC2- containing CM (50, 100%). g In vitro migration was determined for 6 h after recombinant STC2 (100 ng/mL) treatment. Migrated cells were visualised by staining with fluorescent phalloidin and DAPI. Scale bar = 50 µm. h Proteolytic activities of MMP-2 and MMP-9 were determined by gelatin zymography after recombinant STC2 (50, 100, 200 ng/mL) or STC2-containing CM (50, 100%).

Article Snippet: Human recombinant STC2 (Cat#9405-SO) was purchased from R&D Systems.

Techniques: Two Tailed Test, Expressing, Zymography, Recombinant, In Vitro, Migration, Staining

Journal: Cell death discovery

Article Title: Stanniocalcin 2 drives malignant transformation of human glioblastoma cells by targeting SNAI2 and Matrix Metalloproteinases.

doi: 10.1038/s41420-022-01090-6

Figure Lengend Snippet: Fig. 5 Secreted STC2 regulates SNAI2 and MMPs through p38 MAPK pathway. a, b LN18 and A172-STC2 cells were treated with small molecule signaling pathway inhibitors (LGK974, 500 nM; LY3214996, 50 nM; Rapamycin, 10 nM; LY294002 1 µM; and SB202190, 10 µM) for 48 h and STC2 expression was detected by real-time PCR (a) and Western blot analysis (b). Means ± SD; n = 3; On-way ANOVA, ***ī< 0.001. c Wild- type A172 cells were co-treated with small molecule signaling pathway inhibitors and recombinant STC2 (100 ng/mL) for 48 h and SNAI2, MMP- 2, and MMP-9 mRNA expressions were determined by real-time PCR. The relative mRNA levels were calculated against vehicle control of each inhibitors. Small molecule inhibitors were added 30 min prior to recombinant STC2 treatment. Means ± SD; n = 3; Student’s two tailed t-test, *p < 0.05, **p < 0.01, ***p < 0.001. d A172 cells were treated with recombinant STC2 (100 ng/mL) or STC2-containing CM (50%) with or without p38 MAPK inhibitor SB202190 for 48 h. SNAI2 protein expression along with phosphorylated p38 (p-p38) and total p38 (t-p38) proteins were determined by Western blot analysis.

Article Snippet: Human recombinant STC2 (Cat#9405-SO) was purchased from R&D Systems.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Recombinant, Control, Two Tailed Test

Journal: Cells

Article Title: Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells

doi: 10.3390/cells11172684

Figure Lengend Snippet: Recombinant HRG binds STC2 and modulates phagocytosis of bioparticles. ( A ) Co-immunoprecipitation of HRG but not inactive HRG with STC2. Recombinant proteins (2 µg each) were separated on SDS-PAGE as individual preparations (loading control) or after mixing and immunoprecipitation (IP) using antibodies against STC2, followed by immunoblotting (IB) as indicated. ( B ) Ratio of HRG (active or inactive) band intensities in the STC2 immunoprecipitate normalized to corresponding active and inactive HRG loading controls. Statistical analysis; Student’s t -test. ( C ) Representative microscope images of U937 monocytes without (left) or with treatment with active HRG (right) in the phagocytosis assay. Green cells have engulfed pH-sensitive fluorescent bioparticles. Scale bar; 50 µm. ( D ) Quantification of phagocytosis efficiency in the different treatment conditions. The proportion of positive (green) phagocytotic U937 cells to all cells per field of vision is shown in relation to the positive cells/total cells in the vitD3 differentiated HRG-treated condition (set to 1). Statistical analysis; Tukey’s multiple comparisons test ( D ). *** p < 0.001; **** p < 0.0001.

Article Snippet: At the start of the experiment, cells were incubated with 10 nM vitD3, recombinant, in-house purified HRG (mouse) at 1 μg/mL (13.3 nM) [ ], and STC2 (mouse) (cat. no. STC2-16118 M, Creative Biomart, Shirley, NY, USA) or inactive HRG protein at equivalent molar concentrations together with sterile green E. coli bioparticles (cat. no. 4616, Essen Bioscience, Ann Arbor, MI, USA) at 33 μg/mL.

Techniques: Recombinant, Immunoprecipitation, SDS Page, Control, Western Blot, Microscopy, Phagocytosis Assay

Journal: Cells

Article Title: Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells

doi: 10.3390/cells11172684

Figure Lengend Snippet: Affinity determination of HRG’s binding to STC2 using QCM. ( A ) The immobilization of STC2 on the QCM LNB sensor surface. ( B ) Sensorgram and kinetic analysis showing chip-immobilized STC2 and binding of HRG at three different concentrations: 50, 100 and 200 nM. Black lines: experimental curves. Red lines: fitted curves. For representative sensorgram shown, three injections per concentration, two independent experiments. ( C ) Sensorgram showing chip-immobilized STC2 and lack of binding of inactive HRG tested at three different concentrations: 50, 100 and 200 nM. For representative sensorgram shown, three injections per concentration, two independent experiments. ( D ) Sensorgram showing chip-immobilized HRG and lack of binding of STC2, tested at three different concentrations: 200 nM, 450 nM and 900 nM. For representative sensorgram shown, two injections per concentration, three independent experiments.

Article Snippet: At the start of the experiment, cells were incubated with 10 nM vitD3, recombinant, in-house purified HRG (mouse) at 1 μg/mL (13.3 nM) [ ], and STC2 (mouse) (cat. no. STC2-16118 M, Creative Biomart, Shirley, NY, USA) or inactive HRG protein at equivalent molar concentrations together with sterile green E. coli bioparticles (cat. no. 4616, Essen Bioscience, Ann Arbor, MI, USA) at 33 μg/mL.

Techniques: Binding Assay, Concentration Assay

Journal: Cells

Article Title: Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells

doi: 10.3390/cells11172684

Figure Lengend Snippet: Binding of HRG and STC2 individually and together to live U937 cells. ( A ) Schematic outline of the experimental setup. Undifferentiated or vitD3 differentiated U937 cells, immobilized on QCM LNB chips with HRG, STC2 or a mix of the two, injected over chip surfaces. ( B ) Real-time qPCR data of CD14 expression normalized to GAPDH on undifferentiated and vitD3 differentiated U937 cells seeded on the QCM chip. Three independent analyses. ( C – F ) Representative sensorgram showing frequency response from injections over undifferentiated ( C , E , G ) and vitD3 differentiated ( D , F , H ) live U937 cells. Black lines: experimental curves. Red lines: fitted curves of the 1:1 interaction model. For representative sensorgrams shown, three injections per concentration, two independent experiments.

Article Snippet: At the start of the experiment, cells were incubated with 10 nM vitD3, recombinant, in-house purified HRG (mouse) at 1 μg/mL (13.3 nM) [ ], and STC2 (mouse) (cat. no. STC2-16118 M, Creative Biomart, Shirley, NY, USA) or inactive HRG protein at equivalent molar concentrations together with sterile green E. coli bioparticles (cat. no. 4616, Essen Bioscience, Ann Arbor, MI, USA) at 33 μg/mL.

Techniques: Binding Assay, Injection, Expressing, Concentration Assay

Journal: Cells

Article Title: Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells

doi: 10.3390/cells11172684

Figure Lengend Snippet: Affinity of HRG for binding to fixed U937 cells. ( A ) Sensorgram showing frequency response to inactive HRG over vitD3 differentiated, fixed U937 cells. ( B ) Sensorgram showing frequency response to four concentrations (125 nM, 250 nM, 500 nM, 1 µM) of STC2 over fixed, undifferentiated (dashed lines) and vitD3 differentiated (straight lines) U937 cells. The mean of two injections is shown. ( C , D ) Sensorgram and kinetic analysis show the binding of HRG at three concentrations (25, 50 and 100 nM) to undifferentiated ( C ) or vitD3 differentiated ( D ), fixed U937 cells. Black lines: experimental curves. Red lines: fitted curves of the 1:1 interaction model. For representative sensorgrams shown, three injections per concentration, three independent experiments. ( E , F ) Sensorgram and kinetic analysis showing frequency response to three concentrations of HRG (25, 50 and 100 nM) to fixed, undifferentiated ( E ) or vitD3 differentiated ( F ) U937 cells after treatment with heparinase. Black lines: experimental curves. Red lines: fitted curves of 1:2 interaction model. For representative sensorgrams shown, three injections per concentration, three independent experiments.

Article Snippet: At the start of the experiment, cells were incubated with 10 nM vitD3, recombinant, in-house purified HRG (mouse) at 1 μg/mL (13.3 nM) [ ], and STC2 (mouse) (cat. no. STC2-16118 M, Creative Biomart, Shirley, NY, USA) or inactive HRG protein at equivalent molar concentrations together with sterile green E. coli bioparticles (cat. no. 4616, Essen Bioscience, Ann Arbor, MI, USA) at 33 μg/mL.

Techniques: Binding Assay, Concentration Assay