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: Genomics, Proteomics & Bioinformatics

Article Title: LCORL and STC2 Variants Increase Body Size and Growth Rate in Cattle and Other Animals

doi: 10.1093/gpbjnl/qzaf025

Figure Lengend Snippet: Selective sweep and functional analysis of STC2 variants A . Detailed plots for Chr20:4,598,655–5,366,902, containing the STC2 locus. The upper row shows zoomed-in Manhattan plots of ln LR for each breed. The lower row shows allele frequency trajectories for the top SNVs in the CLUES analysis and the missense SNVs. Gray shading indicates the time range from 1000 years ago to the present. B . Stacked bar plot of the haplotypes comprising rs110540352 and rs42661323 in the Hereford cattle ( n = 200 haplotypes). The two derived alleles (rs110540352*G and rs42661323*G) are almost always inherited together as a single haplotype. The y-axis represents the number of haplotypes, and the x-axis represents the indicated SNVs. C . rs42661323 is the lead GWAS SNV for yearling weight and average daily gain in cattle. The GWAS data source is the same as in Figure 2C and . D . Conservation analysis of the amino acid residue at position 60 of STC2. The amino acid residues at positions 60–66 of STC2 interact with PAPP-A, marked with blue shading.

Article Snippet: We commissioned Cyagen Biosciences Inc. to generate the STC2 A60P mice via CRISPR/Cas9-mediated gene editing.

Techniques: Functional Assay, Derivative Assay, Residue

Journal: Genomics, Proteomics & Bioinformatics

Article Title: LCORL and STC2 Variants Increase Body Size and Growth Rate in Cattle and Other Animals

doi: 10.1093/gpbjnl/qzaf025

Figure Lengend Snippet: STC2 A60P increases body weight and body length in mice A . Total body weight of indicated littermates from 3 to 9 weeks of age. Number of male mice in each genotype: Stc2 +/+ ( n = 12), Stc2 A60P/+ ( n = 40), and Stc2 A60P/A60P ( n = 17). Number of female mice in each genotype: Stc2 +/+ ( n = 12), Stc2 A60P/+ ( n = 30), and Stc2 A60P/A60P ( n = 13). Error bars represent mean ± SE. Significant difference in body weight between Stc2 A60P/A60P and Stc2 +/+ mice at each time point was determined by two-sided Student’s t -test (notations above each time point). Significant difference in body weight between the three genotypes of male mice (22 to 63 days) or female mice (22 to 63 days) was determined by two-way ANOVA followed by Tukey’s post hoc test (notations on the far right). B . Body length of Stc2 +/+ , Stc2 A60P/+ , Stc2 A60P/A60P mice at 14 weeks of age. Number of male mice in each genotype: Stc2 +/+ ( n = 12), Stc2 A60P/+ ( n = 40), and Stc2 A60P/A60P ( n = 17). Number of female mice in each genotype: Stc2 +/+ ( n = 12), Stc2 A60P/+ ( n = 30), and Stc2 A60P/A60P ( n = 13). Error bars represent mean ± SE. Significant difference was determined by one-way ANOVA followed by Tukey’s post hoc test. C . Representative images of littermates of Stc2 +/+ , Stc2 A60P/+ , and Stc2 A60P/A60P mice. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ns, not significant.

Article Snippet: We commissioned Cyagen Biosciences Inc. to generate the STC2 A60P mice via CRISPR/Cas9-mediated gene editing.

Techniques:

Journal: Genomics, Proteomics & Bioinformatics

Article Title: LCORL and STC2 Variants Increase Body Size and Growth Rate in Cattle and Other Animals

doi: 10.1093/gpbjnl/qzaf025

Figure Lengend Snippet: Distribution and evolution of LCORL and STC2 variants in cattle A . Geographical distribution of erived alleles at the LCORL (rs384548488*A) and STC2 (rs110540352*G and rs42661323*G) loci. Blue and orange indicate the derived and ancestral alleles, respectively. Half-orange and half-blue triangles and circles indicate the heterozygous ancient and modern cattle, respectively. The modern cattle data are from Run 9 of the 1000 Bull Genomes Project. B . Genotypes of LCORL and STC2 loci in ancient cattle. Triangles indicate the casual variants. Missing genotypes are colored in gray, homozygotes for ancestral allele are colored in light yellow, heterozygotes are colored in orange, and homozygotes for derived allele are colored in dark red.

Article Snippet: We commissioned Cyagen Biosciences Inc. to generate the STC2 A60P mice via CRISPR/Cas9-mediated gene editing.

Techniques: Derivative Assay

Journal: Neoplasma

Article Title: Blocking stanniocalcin 2 reduces sunitinib resistance in clear cell renal cell carcinoma.

doi: 10.4149/neo_2021_210823N1206

Figure Lengend Snippet: Figure 1. Expression pattern of STC2 in KIRC specimens and ccRCC cell lines. A) GEPIA analysis shows the mRNA expression levels of STC2 in 523 KIRCs and 100 non-KIRCs. B, C) The relative expression levels of STC2 in HK-2 cells and ccRCC cell lines were evalu ated by RT-qPCR and western blotting. β-actin was used as a loading reference. D) The STC2 contents in the culture medium of HK-2 cells and ccRCC cell lines were detected by ELISA. *p<0.05, **p<0.01

Article Snippet: Human STC2 neutralization antibody (AF2830), recombinant human STC2 (O76061), and STC2 ELISA Kit (CSB-EL022822HU) were purchased from R&D system (USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Neoplasma

Article Title: Blocking stanniocalcin 2 reduces sunitinib resistance in clear cell renal cell carcinoma.

doi: 10.4149/neo_2021_210823N1206

Figure Lengend Snippet: Figure 3. Effect of STC2 on sunitinib resistance in ccRCC cells. A–C) Determi nation of cell viability of Caki-1, 786-O, and 769-P cells in the absence (Ctrl) or presence of 5 μM sunitinib or 5 μM sunitinib+0.2 μg/ml STC2 neutralizing an tibody. D–F) Determination of cell viability of Caki-1, 786-O, and 769-P cells in the absence (Ctrl) or presence of 5 μM sunitinib or 5 μM sunitinib+500 ng/ ml hSTC2. *p<0.05

Article Snippet: Human STC2 neutralization antibody (AF2830), recombinant human STC2 (O76061), and STC2 ELISA Kit (CSB-EL022822HU) were purchased from R&D system (USA).

Techniques:

Journal: Neoplasma

Article Title: Blocking stanniocalcin 2 reduces sunitinib resistance in clear cell renal cell carcinoma.

doi: 10.4149/neo_2021_210823N1206

Figure Lengend Snippet: Figure 6. Effects of the STC2 neutralizing antibody on sunitinib accumulation, lysosomal pH, cell proliferation, and apoptosis in Caki-1 cells. A) Phase-contrast microscopy showing accumulation of yellow granules in Caki-1 cells incubated in the absence (Ctrl) or presence of sunitinib (5 μM), 5 μM sunitinib+0.2 μg/ml STC2 neutralizing antibody for 24 h. B, C) AO staining indicates the lysosome pH in Caki-1 cells incubated in the absence (Ctrl) or presence of sunitinib (5 μM), 5 μM sunitinib+0.2 μg/ml STC2 neutralizing antibody for 24 h. Quantification of the ratio between the red and green signal of AO was performed by the software ImageJ. D) The fluorescence of the lysosomal probe (LysoTracker™ Green DND-26) in Caki-1 cells incubated in the absence (Ctrl) or presence of sunitinib (5 μM), 5 μM sunitinib+0.2 μg/ml STC2 neutralizing antibody for 24 h. E, F) The immuno fluorescence staining of cell proliferation marker Ki-67 and cell apoptosis detection by TUNEL staining in Caki-1 cells incubated in the absence (Ctrl) or presence of sunitinib (5 μM), 5 μM sunitinib+0.2 μg/ml STC2 neutralizing antibody for 24 h. Quantification of Ki-67 and TUNEL fluorescence was performed by the software ImageJ. NS: no significant difference between control with treatments. *p<0.05

Article Snippet: Human STC2 neutralization antibody (AF2830), recombinant human STC2 (O76061), and STC2 ELISA Kit (CSB-EL022822HU) were purchased from R&D system (USA).

Techniques: Microscopy, Incubation, Staining, Software, Fluorescence, Marker, TUNEL Assay, Control

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

Journal: Metabolites

Article Title: Impact of Long-Term HFD Intake on the Peripheral and Central IGF System in Male and Female Mice

doi: 10.3390/metabo10110462

Figure Lengend Snippet: List of TaqMan probes used for qPCR.

Article Snippet: Stanniocalcin-2 , Stc2 , Mm00441560_m1 , Applied Biosystems.

Techniques: Clinical Proteomics

Journal: PLoS ONE

Article Title: ER stress activation in the intestinal mucosa but not in mesenteric adipose tissue is associated with inflammation in Crohn’s disease patients

doi: 10.1371/journal.pone.0223105

Figure Lengend Snippet: mRNA levels (qRT-PCR) of ATF3 (A), STC2 (B) and DDIT3 (C) were investigated in intestinal mucosa of CD patients (CD Group) compared to controls (CTR Group). mRNA levels (qRT-PCR) of ATF3 (D), STC2 (E) and DDIT3 (F) were evaluated in MAT of CD patients (CD Group) compared to controls (CTR Group). * p < 0.05 and ** p < 0.01 are considered statistically significant versus control group. AU: arbitrary unit.

Article Snippet: Taqman Primers used was: EIF2AK3 (Hs_00178128_m1), ATF3 (Hs_00231069_m1), ATF6 (Hs_00232586_m1), calreticulin (Hs_00189032_m1), STC2 (Hs_00175027_m1), DNAJC3 (Hs_00534483_m1), DDIT3 (Hs_00358796_g1), ERN1 (Hs_00980095_m1), HSPA5 (Hs_99999174_m1), HSP90B1 (Hs_00427665_g1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4326317E) were obtained from Applied Biosystems.

Techniques: Quantitative RT-PCR, Control

Journal: Nature Communications

Article Title: Electrical modulation of transplanted stem cells improves functional recovery in a rodent model of stroke

doi: 10.1038/s41467-022-29017-w

Figure Lengend Snippet: a Heatmap of transcriptome changes in NPCs from electrical stimulation (NPC – unstimulated, NPC Stim – electrically stimulated; blue-downregulated, orange-upregulated). b Volcano plot demonstrating changes in genes with electrical stimulation (blue-downregulated, orange-upregulated). c Top Gene set enrichment pathways with STC2 as the leading edge. d qRT-PCR analysis indicated that STC2 in NPCs was upregulated by electrical stimulation. e ELISA study indicated that the level of STC2 protein after electrical stimulation (NPC Stim ) is much higher than that in non-stimulated NPCs (NPC). d , e Analyzed using a one-way ANOVA, followed by Tukey’s HSD post-hoc test with ** P < 0.01, **** P < 0.0001, data shown as mean ± SEM, n = 4.

Article Snippet: For STC2 gain and loss of function, hNPCs were treated with STC2 lentiviral activation particles (sc-405292-LAC) and STC2 shRNA (h) particles (sc-44127-V) respectively as per the manufacturer protocol (Santa Cruz Biotechnology Inc., Lentiviral activation particles transduction).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Electrical modulation of transplanted stem cells improves functional recovery in a rodent model of stroke

doi: 10.1038/s41467-022-29017-w

Figure Lengend Snippet: a Vibrissae-forepaw (WP) and total neurological score (NS). There is a statistically significant difference in NPC Stim , STC2 UP , and Scramble KD+Stim groups versus all other groups starting at week 3 and onward for WP Scores and week 5 and onward for NS scores. STC2 KD and STC2 KD+Stim showed no significant behavioral recovery at any timepoint in either WP Scores or NS Scores. b Representative images of fluorescently labeled cells. Green indicates BrdU positive cells, whereas Blue indicates cell nucleus. Scale bar indicates 200 µm. c BrdU positive cells in certain areas of interest. Data are shown as mean ± SEM, n = 8 images/4 rats. d PAX6/BrdU or Nestin/BrdU positive cells in certain areas of interest. Data are shown as mean ± SEM, n = 16 images/4 rats. e Representative images of fluorescently labeled cells. Green indicates Nestin positive cells whereas Red indicates PAX6 positive cells. Blue indicates BrdU positive cells. Scale bar indicates 200 µm. a Analyzed using a Kruskal–Wallis test followed by post-hoc pairwise Mann–Whitney U test with Benjamini–Hochberg correction to control the false discovery rate at the 0.05 level. For both, * P < 0.01, ** P < 0.001, data shown as mean ± SEM, n = 10 per group. c , d Analyzed using a one-way ANOVA, followed by Tukey’s HSD post-hoc test with * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: For STC2 gain and loss of function, hNPCs were treated with STC2 lentiviral activation particles (sc-405292-LAC) and STC2 shRNA (h) particles (sc-44127-V) respectively as per the manufacturer protocol (Santa Cruz Biotechnology Inc., Lentiviral activation particles transduction).

Techniques: Labeling, MANN-WHITNEY, Control

Journal: Nature Communications

Article Title: Electrical modulation of transplanted stem cells improves functional recovery in a rodent model of stroke

doi: 10.1038/s41467-022-29017-w

Figure Lengend Snippet: a Schematic of the brain slice view (left) and top view (right) in the skull for STC2 protein delivery via mini-osmotic pump. The cannula of the osmotic pump is implanted at 0.8 mm posterior (Y) and 1.5 mm contralateral (X) to the bregma at a depth of 3.5 mm at 1-week after stroke. b Behavioral testing. Vibrissae-forepaw (WP) behavioral testing (left) and overall neurological score (right). Based on WP, STC2 group exhibited statistically significantly greater recovery beginning at 4 weeks post-stroke compared to other groups. c Endogenous neuroblasts. Representative images of DCX + /BrdU+ cells (left) and the cell count of number co-positive cells (right) at peri-infarct region. b Analyzed using a log-transform two-way repeated measures ANOVA which revealed a statistically significant interaction between the effects of treatment group and WP Score ( F [12, 162] = 7.058, P < 0.0001); followed by Dunnett’s multiple comparisons test. Neurological scores (NS) were analyzed using a Kruskal–Wallis test ( P = 0.018), followed by Dunn’s multiple comparisons test. For both, * P < 0.05, ** P < 0.01, data shown as mean ± SEM, n = 10 per group. c Analyzed using one-way ANOVA, followed by Tukey’s HSD post-hoc test with * P < 0.05. Data shown as mean ± SEM, n = 20 images/4 rats.

Article Snippet: For STC2 gain and loss of function, hNPCs were treated with STC2 lentiviral activation particles (sc-405292-LAC) and STC2 shRNA (h) particles (sc-44127-V) respectively as per the manufacturer protocol (Santa Cruz Biotechnology Inc., Lentiviral activation particles transduction).

Techniques: Slice Preparation, Cell Counting