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: 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: The human STC2 expression plasmid (Cat#RC200537, OriGene) and shRNA plasmid kit (Locus ID 8614, Cat#TR309053, OriGene) were transfected using Xfect (Takara Bio Company according to the manufacturer’s protocol.

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: The human STC2 expression plasmid (Cat#RC200537, OriGene) and shRNA plasmid kit (Locus ID 8614, Cat#TR309053, OriGene) were transfected using Xfect (Takara Bio Company according to the manufacturer’s protocol.

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: The human STC2 expression plasmid (Cat#RC200537, OriGene) and shRNA plasmid kit (Locus ID 8614, Cat#TR309053, OriGene) were transfected using Xfect (Takara Bio Company according to the manufacturer’s protocol.

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: The human STC2 expression plasmid (Cat#RC200537, OriGene) and shRNA plasmid kit (Locus ID 8614, Cat#TR309053, OriGene) were transfected using Xfect (Takara Bio Company according to the manufacturer’s protocol.

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: The human STC2 expression plasmid (Cat#RC200537, OriGene) and shRNA plasmid kit (Locus ID 8614, Cat#TR309053, OriGene) were transfected using Xfect (Takara Bio Company according to the manufacturer’s protocol.

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