Journal: Life Science Alliance

Article Title: Identification and characterization of a membrane receptor that binds to human STC1

doi: 10.26508/lsa.202201497

Figure Lengend Snippet: (A) hSTC1, (B) hSTC2, and (C) hCREG binding to human IGF2R.

Article Snippet: To perform kinetic affinity assays, hSTC1-His, human His-tagged STC2 (hSTC2-His, 9405-SO-050; R&D Systems), and human His-tagged cellular repressor of E1A-stimulated gene (hCREG-His, 2380-CR-025/CF; R&D Systems) were prepared at 10 μg/ml in a running buffer, followed by a twofold serial dilution to give 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, and 0.019 μg/ml solutions.

Techniques: Binding 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: Molecular medicine reports

Article Title: Stanniocalcin 2 improved osteoblast differentiation via phosphorylation of ERK.

doi: 10.3892/mmr.2016.5951

Figure Lengend Snippet: Figure 1. Knockdown of STC2 reduced osteoblast differentiation and mineralization. (A) Protein expression levels of STC2 in C2C12 and MC3T3‑E1 cells. (B) STC2 protein expression levels during differentiation of MC3T3‑E1 cells to osteoblasts. (C) Knockdown of STC2 in MC3T3‑E1 cells. (D) ALP activity. *P<0.05 vs. the SCR group. (E) ALP staining following induction of osteoblast differentiation for 6 days. (F) Alizarin red S staining for detection of mineral ized matrix formation following induction of osteoblast differentiation for 14 days. STC2, stanniocalcin 2; SCR, scramble; shSTC2, short hairpin STC2; ALP, alkaline phosphatase.

Article Snippet: Following blocking with milk, membranes were incubated with primary antibodies against STC2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-14350), phosphorylated (p)-ERK1/2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 9102) and β-actin (1:3,000; Beyotime Institute of Biotechnology, Haimen, China; cat. no. AF0003) overnight at 4 ̊C, followed by incubation with horseradish peroxidase (HRP)-labeled anti-mouse (cat. no. 7076) or anti-rabbit (cat. no. 7074) IgG secondary antibodies (1:2,000; Cell Signaling Technology, Inc.) for 1 h at room temperature.

Techniques: Knockdown, Expressing, Activity Assay, Staining

Journal: Molecular medicine reports

Article Title: Stanniocalcin 2 improved osteoblast differentiation via phosphorylation of ERK.

doi: 10.3892/mmr.2016.5951

Figure Lengend Snippet: Figure 2. Knockdown of STC2 reduced osteoblast‑specific gene expression of (A) Runx2, (B) Col1α1, (C) OSX and (D) OCN. *P<0.01 vs. the scramble group. STC2, stanniocalcin 2; shSTC2, short hairpin STC2; Runx2, runt‑related transcription factor 2; Col1α1, collagen type I α 1 chain; OSX, osterix; OCN, osteocalcin.

Article Snippet: Following blocking with milk, membranes were incubated with primary antibodies against STC2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-14350), phosphorylated (p)-ERK1/2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 9102) and β-actin (1:3,000; Beyotime Institute of Biotechnology, Haimen, China; cat. no. AF0003) overnight at 4 ̊C, followed by incubation with horseradish peroxidase (HRP)-labeled anti-mouse (cat. no. 7076) or anti-rabbit (cat. no. 7074) IgG secondary antibodies (1:2,000; Cell Signaling Technology, Inc.) for 1 h at room temperature.

Techniques: Knockdown, Gene Expression

Journal: Molecular medicine reports

Article Title: Stanniocalcin 2 improved osteoblast differentiation via phosphorylation of ERK.

doi: 10.3892/mmr.2016.5951

Figure Lengend Snippet: Figure 3. Overexpression of STC2 increased osteoblast differentiation and mineralization. (A) Overexpression of STC2 in MC3T3‑E1 cells. (B) ALP activity, *P<0.05 vs. the Ctrl. (C) ALP staining following induction of osteoblast differentiation for 6 days. (D) Alizarin red S staining for detection of mineralized matrix formation following induction of osteoblast differentiation for 14 days. STC2, stanniocalcin 2; Ctrl, control; ALP, alkaline phosphatase.

Article Snippet: Following blocking with milk, membranes were incubated with primary antibodies against STC2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-14350), phosphorylated (p)-ERK1/2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 9102) and β-actin (1:3,000; Beyotime Institute of Biotechnology, Haimen, China; cat. no. AF0003) overnight at 4 ̊C, followed by incubation with horseradish peroxidase (HRP)-labeled anti-mouse (cat. no. 7076) or anti-rabbit (cat. no. 7074) IgG secondary antibodies (1:2,000; Cell Signaling Technology, Inc.) for 1 h at room temperature.

Techniques: Over Expression, Activity Assay, Staining, Control

Journal: Molecular medicine reports

Article Title: Stanniocalcin 2 improved osteoblast differentiation via phosphorylation of ERK.

doi: 10.3892/mmr.2016.5951

Figure Lengend Snippet: Figure 5. STC2 regulated ERK phosphorylation. (A) ERK phosphorylation was reduced when STC2 expression was downregulated. (B) ERK phosphorylation was increased when STC2 was overexpressed. STC2, stanniocalcin 2; shSTC2, short hairpin STC2; p‑ERK, phosphorylated‑extracellular signal‑regulated kinase.

Article Snippet: Following blocking with milk, membranes were incubated with primary antibodies against STC2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-14350), phosphorylated (p)-ERK1/2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 9102) and β-actin (1:3,000; Beyotime Institute of Biotechnology, Haimen, China; cat. no. AF0003) overnight at 4 ̊C, followed by incubation with horseradish peroxidase (HRP)-labeled anti-mouse (cat. no. 7076) or anti-rabbit (cat. no. 7074) IgG secondary antibodies (1:2,000; Cell Signaling Technology, Inc.) for 1 h at room temperature.

Techniques: Phospho-proteomics, Expressing

Journal: Molecular medicine reports

Article Title: Stanniocalcin 2 improved osteoblast differentiation via phosphorylation of ERK.

doi: 10.3892/mmr.2016.5951

Figure Lengend Snippet: Figure 4. Overexpression of STC2 increased osteoblast specific gene expression of (A) Runx2, (B) Col1α1, (C) OSX and (D) OCN. *P<0.01 vs. the Ctrl group. STC2, stanniocalcin 2; Runx2, runt‑related transcription factor 2; Col1α1, collagen type I α 1 chain; OSX, osterix; OCN, osteocalcin; Ctrl, control.

Article Snippet: Following blocking with milk, membranes were incubated with primary antibodies against STC2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-14350), phosphorylated (p)-ERK1/2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 9102) and β-actin (1:3,000; Beyotime Institute of Biotechnology, Haimen, China; cat. no. AF0003) overnight at 4 ̊C, followed by incubation with horseradish peroxidase (HRP)-labeled anti-mouse (cat. no. 7076) or anti-rabbit (cat. no. 7074) IgG secondary antibodies (1:2,000; Cell Signaling Technology, Inc.) for 1 h at room temperature.

Techniques: Over Expression, Gene Expression, Control

Journal: Molecular medicine reports

Article Title: Stanniocalcin 2 improved osteoblast differentiation via phosphorylation of ERK.

doi: 10.3892/mmr.2016.5951

Figure Lengend Snippet: Figure 6. Inhibition of ERK phosphorylation altered osteoblast‑specific gene expression of (A) Runx2. (B) Col1α1. (C) OSX and (D) OCN stimulated by STC2 overexpression. Cells are treated with U0126 at a final concentration of 20 µM. *P<0.01 vs. the Ctrl group. STC2, stanniocalcin 2; Runx2, runt‑related transcription factor 2; Col1α1, collagen type I α 1 chain; OSX, osterix; OCN, osteocalcin.

Article Snippet: Following blocking with milk, membranes were incubated with primary antibodies against STC2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-14350), phosphorylated (p)-ERK1/2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 9102) and β-actin (1:3,000; Beyotime Institute of Biotechnology, Haimen, China; cat. no. AF0003) overnight at 4 ̊C, followed by incubation with horseradish peroxidase (HRP)-labeled anti-mouse (cat. no. 7076) or anti-rabbit (cat. no. 7074) IgG secondary antibodies (1:2,000; Cell Signaling Technology, Inc.) for 1 h at room temperature.

Techniques: Inhibition, Phospho-proteomics, Gene Expression, Over Expression, Concentration 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 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

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: ACS Applied Materials & Interfaces

Article Title: 3D Culture Modeling of Metastatic Breast Cancer Cells in Additive Manufactured Scaffolds

doi: 10.1021/acsami.2c07492

Figure Lengend Snippet: Expression of dormancy-related markers in MDA-MB-231 cells cultured in different configurations. (a) WB corresponding to the intracellular STC1 expression of MDA-MB-231 cells cultured for 8 days on either 2D TCP or 3D PA scaffolds. α-Tubulin was used as a loading control. (b) Bar graph corresponding to normalized to cell number STC1 secretion, measured by ELISA from the supernatant of cell cultures at t = 10 days. (c) Bar graph corresponding to normalized to cell number STC2 secretion measured by ELISA from the supernatant of cell cultures at t = 10 days. (d) Bar graph representing WB semiquantitative analysis of STC1 expression corresponding to normalized (to α-tubulin) STC1 expression after 8 days of cell culture on the various systems. (e) Average cell density corresponding to each culture condition of STC1 experiments. The cell number of each scaffold (data not shown) was used for the STC1 normalization corresponding to each scaffold. (f) Average cell density corresponding to each culture condition of STC2 experiments. The cell number of each scaffold was used for the STC2 normalization of each scaffold. Biological triplicates were used per experiment, and technical triplicates were used during the performance. Error bars represent SD, and * and ** denote P < 0.05 and P < 0.005, respectively.

Article Snippet: Samples were centrifuged for 15 min at 4 °C at 1000 rcf, and their supernatants were assessed using the STC1 and STC2 ELISA kits provided by Cusabio (CSB-EL022821HU) and EIAAB (E12206h), respectively.

Techniques: Expressing, Cell Culture, Control, Enzyme-linked Immunosorbent Assay

Journal: ACS Applied Materials & Interfaces

Article Title: 3D Culture Modeling of Metastatic Breast Cancer Cells in Additive Manufactured Scaffolds

doi: 10.1021/acsami.2c07492

Figure Lengend Snippet: Cell plasticity of MDA-MB-231 cells precultured in different cell culture configurations. (a) WB corresponding to the intracellular STC1 expression of “primed” MDA-MB-231 cells further cultured for 2 days on TCP under the same cell densities. α-Tubulin was used as a loading control. (b) Bar graph corresponding to secreted STC1 normalized to cell number. STC1 is measured by ELISA from the supernatant of “primed” MDA-MB-231 cells after 2 days of culture on TCP under the same cell densities and normalized to the cell number shown in the (e) bar graph. (c) Bar graph corresponding to STC2 secretion normalized to cell number. STC2 is measured by ELISA from the supernatant of “primed” MDA-MB-231 cells after 2 days of culture on TCP under the same cell densities and normalized to the cell number shown in the (f) bar graph. (d) Bar graph representing WB semiquantitative analysis of STC1 expression corresponding to STC1 expression after 2 days of cell culture and normalized to α-tubulin. (e) Average cell number determined by DNA quantification corresponding to each culture condition of STC1 experiments. (f) Average cell number determined by DNA quantification corresponding to each culture condition of STC2 experiments. Biological triplicates were used per experiment, and technical triplicates were used during the performance of ELISAs. Error bars represent SD. One-way ANOVA shows no statistical differences among all groups of “primed” cells once cultured for 2 additional days on 2D TCP. One-way ANOVA shows no statistical differences of cell number among all groups in the bar graph (e) corresponding to STC1 secretion experiments and the bar graph (f) corresponding to STC2 secretion experiments.

Article Snippet: Samples were centrifuged for 15 min at 4 °C at 1000 rcf, and their supernatants were assessed using the STC1 and STC2 ELISA kits provided by Cusabio (CSB-EL022821HU) and EIAAB (E12206h), respectively.

Techniques: Cell Culture, Expressing, Control, 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 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