Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: The expression of SMARCAD1 is significantly higher in pancreatic cancer and positively correlated with poor prognosis. A. The mRNA expression level of SMARCAD1 in pancreatic cancer is much higher than that of normal tissues from GSE16515, GSE11838 and GSE15471. B-C. Expression levels of SMARCAD1 by immunohistochemistry performed with tissue microarray of PC (n=69) and adjacent normal tissues (n=68). Representative images showed positive expression of SMARCAD1 in PC and negative expression in paired normal tissues, respectively. Scale bars=50μm. D. Kaplan-Meier analysis shows the correlation between SMARCAD1 expression and overall survival in patients. Patients with high SMARCAD1 expression had poorer overall survival than those with low expression. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 enhances proliferation of PANC-1 cells. A-B. The efficiency of SMARCAD1 knockdown (A) or overexpression (B) in PANC-1 cells was detected by western blotting. β-actin was used as an internal control. C-D. CCK8 assay was performed to determine the proliferation of PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) at the indicated time points after plated. Cell viability was measured at 450nm. E-F. The effect of SMARCAD1 knockdown (E) or overexpression (F) on Colony-forming of PANC-1 cells was shown in the top panels. Number of foci was counted as shown in the bottom panels. All data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Knockdown, Over Expression, Western Blot, Control, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 promotes migration and invasion of PANC-1 cells. A-B. Effect of SMARCAD1 knockdown (A) or SMARCAD1 overexpression (B) on cell migration was detected by wound healing at indicated time points after scratching. The wound healing was measured by ImageJ software. C-D. Motility ability of PANC-1 cells with SMARCAD1 depletion (C) or overexpression (D) was assessed by transwell assay at 24h. Representative images of migration were photographed at 24h (Top panel). The number of migrated cells was counted from 5 randomly selected fields under microscope (Bottom panel). E-F. Invasion ability of PANC-1 cells with SMARCAD1 depletion (E) or overexpression (F) was assessed by transwell assay at 48h. Representative images of invasion were photographed at 48h (Top panel). The number of invaded cells was counted from 5 randomly selected fields under microscope (Bottom panel). Scale bars=150um. Data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Migration, Knockdown, Over Expression, Software, Transwell Assay, Microscopy

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 induces EMT in PANC-1 cells. A-B. The morphology changes of PANC-1 cells: cells lose contact with each other with SMARCAD1 depletion (A) or gain more contact with SMARCAD1 overexpression (B), Scale bars=250μm. C-D. Changes in mRNA level of EMT relative markers were tested by Quantitative real-time PCR in SMARCAD1 knockdown (C) or overexpression (D) cells. The results were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. (D). E-F. The protein levels of EMT relative markers in SMARCAD1 knockdown (E) or overexpression (F) cells were assessed by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Knockdown, Control, Western Blot

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1-induced EMT was regulated by Wnt/beta-catenin signaling pathway. A-B. The mRNA level of β-catenin was detected by Quantitative real-time PCR in PANC-1 cells with SMARCAD1 knockdown (A) or overexpression (B) respectively. The data were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. C-D. β-catenin, cyclin-D1, c-Myc and survivin protein levels were assayed by western blotting in PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) respectively. E. PANC-1 cells with SMARCAD1 depletion were treated with CHIR99021 (6μM/ml) for 24h. The protein levels of EMT markers and Wnt/β-catenin target genes (β-catenin, cyclin-D1, c-Myc and survivin) were detected by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Over Expression, Control, Western Blot

Journal: Journal of Toxicologic Pathology

Article Title: Characteristic Upregulation of Glucose-Regulated Protein 78 in an Early Lesion Negative for Hitherto Established Cytochemical Markers in Rat Hepatocarcinogenesis

doi: 10.1293/tox.22.281

Figure Lengend Snippet: The relative expression of GRP78 mRNA in GST-P-negative HAF induced by DEN→clofibrate was slightly increased compared with the adjacent normal tissue. The vertical axis represents the ratio of GRP78 mRNA expression versus GAPDH mRNA expression in each region. The white bars represent GRP78 mRNA expression in the normal tissue, and the left hatched and black bars represent GST-P-positive and GST-P-negative HAF, respectively.

Article Snippet: That for GRP78 was accomplished in accordance with the protocol provided in the CSA II Kit (Dako Japan, Tokyo), with a minor modification consisting of retrieval of antigens using Target Retrieval Solution (Dako Japan), using a primary goat anti-GRP78 antibody (1:1000, room temperature, 15 min, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Journal of Toxicologic Pathology

Article Title: Characteristic Upregulation of Glucose-Regulated Protein 78 in an Early Lesion Negative for Hitherto Established Cytochemical Markers in Rat Hepatocarcinogenesis

doi: 10.1293/tox.22.281

Figure Lengend Snippet: Immunohistochemistry for GRP78 and GST-P in HAF induced by DEN→clofibrate. (a) GST-P-negative lesion: 1, HE staining; 2, GRP78; 3, GST-P. (b) GST-P positive lesion: 1, HE staining; 2, GRP78; 3, GST-P. Lens × 4.

Article Snippet: That for GRP78 was accomplished in accordance with the protocol provided in the CSA II Kit (Dako Japan, Tokyo), with a minor modification consisting of retrieval of antigens using Target Retrieval Solution (Dako Japan), using a primary goat anti-GRP78 antibody (1:1000, room temperature, 15 min, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Immunohistochemistry, Staining

Journal: Journal of Toxicologic Pathology

Article Title: Characteristic Upregulation of Glucose-Regulated Protein 78 in an Early Lesion Negative for Hitherto Established Cytochemical Markers in Rat Hepatocarcinogenesis

doi: 10.1293/tox.22.281

Figure Lengend Snippet: Immunohistochemistry for GRP78 in GST-P-negative HCAs induced by DEN→clofibrate. (a) HE staining. (b) GST-P. (c), (d) GRP78. (a)-(c) lens × 4; (d) lens × 10.

Article Snippet: That for GRP78 was accomplished in accordance with the protocol provided in the CSA II Kit (Dako Japan, Tokyo), with a minor modification consisting of retrieval of antigens using Target Retrieval Solution (Dako Japan), using a primary goat anti-GRP78 antibody (1:1000, room temperature, 15 min, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Immunohistochemistry, Staining

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: qRT-PCR primers

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: ID1 expression was induced by etoposide in ESCC cell lines. A, up-regulated ID1 mRNA level was detected in 34 tumors compared with normal adjacent epithelia by qRT-PCR (paired t test). B, an example case showed the expression of ID1 in ESCC tumors and normal counterparts by immunohistochemistry staining on the tissue microarray (upper panels). Quantitative analysis of the ID1 staining between ESCC tissues and the matched normal esophageal epithelia is shown in the lower panel (paired t test). C, mRNA and protein level of endogenous ID1 was detected in ESCC cell lines by qRT-PCR (left panel) and Western blot (right panel). D, KYSE140, KYSE150, and KYSE450 cells were treated with 10 μm etoposide for the indicated time and harvested. ID1 expression was determined by qRT-PCR (upper panels) and Western blot (lower panels). β-Actin was used as a loading control. The data are shown as means ± S.E. from multiple independent experiments, one-way analysis of variance test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Microarray, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: Overexpression of ID1 enhances cellular resistance to etoposide. A, KYSE150, KYSE140, KYSE450, and KYSE180 cells were treated with increasing concentrations of etoposide for 48 h, and then cell viability was examined by MTS assay. B, KYSE150 and KYSE450 cells stably transfected with empty vector (pLVX) or ID1 (pLVX-ID1) were incubated with DMSO (control) or etoposide (10 μm) for the indicated time, and cell growth was detected using MTS assay. The values are the means ± S.D. of absorbance at 490 nm for three independent experiments. C and D, ID1 transfectants and empty vector controls were treated with 10 μm etoposide for 48 h and then subjected to annexin V-FITC and propidium iodide (PI) staining. The values are expressed as percentages of annexin V-positive versus total cells (C). The expression levels of ID1, p53, cleaved caspase 3, and PARP were examined by Western blot (D). β-Actin was used as a loading control. E and F, KYSE450 cells were transiently transfected with negative control (NC) or ID1 siRNA, followed by 10 μm etoposide treatment. The cells were labeled with annexin V-FITC and propidium iodide and analyzed by flow cytometry. The values are expressed as percentages of annexin V-positive versus total cells (E). The expression levels of ID1, p53, cleaved caspase 3, and PARP were examined by Western blot (F). β-Actin was used as a loading control. The data are expressed as means ± S.D. *, p < 0.05; **, p < 0.01, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Over Expression, MTS Assay, Stable Transfection, Transfection, Plasmid Preparation, Incubation, Control, Staining, Expressing, Western Blot, Negative Control, Labeling, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: Up-regulating ID1 upon etoposide activation is mediated through AP-1 binding sites. A, KYSE450 cells were transiently transfected with the promoter construct of ID1 for 24 h and treated with 10 or 20 μm etoposide. After 24 h, the luciferase activity was determined and normalized to an internal cytomegalovirus Renilla luciferase control. B, comparison of nucleotide sequences among seven different species. The AP-1 DNA binding site is represented with a shaded box. * indicates the same nucleotide sequence. C, schematic representation depicts the location of the mutant variant in the 2-kb ID1 promoter. TSS stands for transcription start site (upper panel). KYSE450 cells were cotransfected with ID1 wild-type or mutant luciferase reporters, together with c-Jun/c-Fos or control vector for 24 h. Then luciferase activity was determined and normalized to an internal cytomegalovirus Renilla luciferase control. The data are shown as means ± S.E. from multiple independent experiments (lower panel). D, KYSE450 cells were transfected with ID1 promoter reporter construct containing either wild-type or mutant putative AP-1 binding site and treated with or without 10 μm etoposide, and then the luciferase activity was determined. E, KYSE450 cells were treated with 10 μm etoposide for 4 h, and then ChIP assays were carried out with antibody against c-Jun, c-Fos, or IgG. The percentages of input of coprecipitating DNAs were calculated by qRT-PCR. The data represent the means ± S.D. of triplicate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Activation Assay, Binding Assay, Transfection, Construct, Luciferase, Activity Assay, Control, Comparison, Sequencing, Mutagenesis, Variant Assay, Plasmid Preparation, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: The activation of ID1 required c-Jun/c-Fos in response to etoposide. A and B, KYSE450 cells were treated with 10 μm etoposide for the indicated time, and the expression of c-Jun/c-Fos and ID1 was determined by qRT-PCR (A) and Western blot (B). C, KYSE450 cells were transiently transfected with c-Jun, c-Fos, c-Jun/c-Fos, and TAM67 as described under “Experimental Procedures.” After 24 h, the expression of c-Jun, c-Fos, and ID1 was determined by qRT-PCR and Western blot. D, KYSE450 and KYSE150 cells were transiently transfected with c-Jun/c-Fos siRNA as described under “Experimental Procedures.” After 24 h, the expression of c-Jun, c-Fos, and ID1 was determined by Western blot. The data represent the means ± S.D. of triplicate experiments. NC, negative control. **, p < 0.01; ***, p < 0.001, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: ID1 inhibits etoposide-induced cell apoptosis in a c-Jun/c-Fos-dependent manner. A, KYSE450 cells were transiently transfected with either negative control (NC) or c-Jun/c-Fos siRNA as indicated. After 24 h, cells were incubated with DMSO or 10 μm etoposide. The expression of c-Jun, c-Fos, ID1, p53, cleaved caspase 3, and PARP was determined by Western blot. β-Actin was used as a loading control. B, KYSE450 cells were transiently transfected with c-Jun/c-Fos siRNA and rescued ID1 with pLVX-ID1 compared with pLVX for 24 h. After that, cells were treated with DMSO or 10 μm etoposide and then subjected to Annexin V-FITC and propidium iodide (PI) staining. The values are expressed as a percentage of annexin V-positive versus total cells. The data are expressed as means ± S.D. *, p < 0.05, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Transfection, Negative Control, Incubation, Expressing, Western Blot, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: Positive correlation between c-Jun/c-Fos and ID1 in human cancers and prognostic value of high c-Jun/c-Fos and ID1 expression for cancer patients survive. A, a statistically significant positive correlation between c-Jun/c-Fos and ID1 mRNA was observed by Pearson's method in ESCC and patients in three independent published data sets including acute myeloid leukemia (GSE12417), ovarian cancer (GSE49997), and colorectal cancer (GSE24551), Pearson correlation analysis. B, clinical outcome data were analyzed by using PROGgeneV2 from published studies for correlations between c-Jun/c-Fos-ID1 expression levels and survival of cancer patients.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Expressing

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 expression is up-regulated in osteosarcoma cells. (A) , mRNA levels of NRP2 in normal osteoblast (NHOst) and osteosarcoma cell lines (Saos-LM7, 143B, 143.98.2, MG-63, MNNG/HOS, Saos-2, and U2-OS) were determined by real time PCR. (B) , the protein levels of NRP2 in NHOst and seven osteosarcoma cell lines were detected by western blot using NRP2 antibody. The relative protein levels were determined by densitometry and normalized with β-actin level. (C) , Kaplan-Meier survival curves of disease–specific mortality for patients whose osteosarcoma expressed or didn’t express NRP2. The log-rank test was used to compare differences between two groups. NRP2 expression was predictive of poor overall survival. Significant difference is indicated by (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. Column: mean value; Error bars: SEM

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 knockdown inhibited both in vitro and in vivo tumor growth. (A & B) , NRP2 expression was knocked down by NRP2 shRNA in 143B cells. Knockdown efficiency was determined by real-time PCR (A) and Western blotting (B) . (C) , By MTT assay, NRP2 knockdown suppressed anchorage-dependent growth of osteosarcoma 143B cells. (D) Soft agar assay. NRP2 knockdown did not reduce the number of colony formed by 143B cells. 143B cells transfected with ShNRP2 formed smaller colony than control vector transfected cells as shown in the representative images of soft agar (insert). (E) & (F) NRP2 knockdown inhibited the in vivo tumor growth in xenograft nude mice model. 1 × 10 6 143B cells were inoculated in NCR nu-nu nude mice. Tumor size was measured every 3 days and a tumor growth curve was created (E) . Points, mean tumor volume; Bars, SEM. (F) , Representative picture of tumor harvested at day 21. (G) , the knockdown of NRP2 expression in tumor samples was confirmed by immunofluorescence staining using NRP2 antibody. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01. Column: mean value; Error bars: SEM.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, In Vitro, In Vivo, Expressing, shRNA, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, Soft Agar Assay, Transfection, Control, Plasmid Preparation, Immunofluorescence, Staining

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: Knockdown of NRP2 inhibited the tumor invasion, migration and lung metastasis of osteosarcoma. (A) , Migration assay. The BD chamber system without Matrigel coating was used to evaluate the migration of shNRP2 and control vector transfected osteosarcoma 143B cells. The migration of 143B cells was significantly inhibited by NRP2 knockdown. (B) , Matrigel invasion assay was performed in BD chamber system coated with Matrigel, using shNRP2 and control vector transfected osteosarcoma 143B cells. There were less NRP2 depleted 143B cells invaded through the matrigel coated porous membrane. (C) , Knockdown of NRP2 in 143B cells reduced the lung metastasis of osteosarcoma in an orthotopic lung metastasis mouse model. Mouse lungs were fixed in Bouin’s solution, and the number of lung surface metastatic nodules was counted and graphed. Each group contained 10 mice and the experiment was repeated 3 times (D) , Representative photographs of lungs with metastatic nodules of osteosarcoma. (E) , NRP2 knockdown significantly reduced both 143B and Saos-2 cells adherence to the endothelial monolayer. The mean cell number was calculated from 10 fields (×100). (F) , Representative images of GFP transfected tumor cells adhering to the endothelial monolayer. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. Column: mean value; Error bars: SEM.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, Migration, Control, Plasmid Preparation, Transfection, Invasion Assay, Membrane

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: Knockdown of NRP2 resulted in decreased blood vessel density of OS in vivo . (A) , Blood vessels (top panel) and capillaries (bottom panel) in tumor samples were visualized by immunohistochemistry with CD31 antibody. Tumor cell nuclear was stained with DAPI. Number of blood vessels per field (100×) was calculated and graphed (Right). Column, mean number of blood vessel per field (x100); Error bars, SEM. (B & C) , Matrigel tube formation assay: no difference was found in the number of tubules formed by HUVEC in conditioned medium from shNRP2 OS cells and shRNA control cells. (B) Representative photographs of tubules formed by HUVEC cells on Matrigel. (C) The tubular number was calculated and graphed, Column: mean number of tubules; Error bars: SEM. (D) , tumor-endothelial co-culture tube formation assay: The HUVEC cells were stained with CellTracker Red CMTPX dye and the tumor cells were transfected with shNRP2 vector with GFP expression. Left panel: tubules formed by HUVEC (red); middle panel: tumor cells (green) attached on tubules; right panel: merged image (right) of HEVEC tubules (red) and attached tumor cells (green). NRP2 depleted tumor cells sustained distinct morphologic changes compared to control cells (bottom panel).

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, In Vivo, Immunohistochemistry, Staining, Tube Formation Assay, shRNA, Control, Co-Culture Assay, Transfection, Plasmid Preparation, Expressing

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 knockdown suppressed endothelial recruitment by osteosarcoma cells. (A) , in a trans-well co-culture migration model, less endothelial cells migrated through the porous insert membrane of the Chamber in shNRP2 group compared to control group. shNRP2 depleted osteosarcoma cells vs control cells were seeded in the lower chamber. HUVECs were seeded in the top insert chamber. Insert picture: Representative photograph of migrated HUVECs. (B) , in a trans-well co-culture invasion model, less endothelial cells invaded through the matrigel coated porous insert membrane of the Chamber in shNRP2 group compared to control group. shNRP2 depleted osteosarcoma cells vs control cells were seeded in the lower chamber. HUVECs were seeded in the top insert chamber. Insert picture: Representative photograph of invaded HUVECs. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01. Column: mean migrated/invaded cells per field (40×); Error bars: SEM.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, Co-Culture Assay, Migration, Membrane, Control

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 expression is regulated by Wnt signaling pathway. A , Genechip® microarray showed the down-regulated expression of NRP2 in Wnt antagonist sLRP5 transfected osteosarcoma Saos-2 cells. VEGF expression is intact. Real time PCR (B) and western blot (C) with accompanying densitometric assay (D) confirmed the down-regulated mRNA and protein level of NRP2 in Wnt antagonist sLRP5 transfected Saos-2 cells, while VEGF level remains intact. E , western blot and accompanying densitometry showed the down-regulated NRP2 expression in additional Wnt antagonist DKK3 transfected Saos-2 cells, 143B cells and U2-OS cells, and Wif-1 transfected 143B cells. F , Chromatin immunoprecipitation (ChIP) assay verified the binding of TCF4 on the five binding sites in NRP2 promoter region. G , schematic representation of the binding sites of TCF/LEF on the 3-kb promoter region of NRP2 genes.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Microarray, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Binding Assay

Journal: BMC Cancer

Article Title: Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer

doi: 10.1186/1471-2407-14-620

Figure Lengend Snippet: TARS mRNA levels are increased in ovarian cancer and reduced by neo - adjuvant chemotherapy. Data were obtained from GEO dataset GDS 2785. The study analyzed mRNA by GeneChip microarray in ovarian tissue from 10 patients with benign conditions, 9 patients with untreated adenocarcinoma, and 24 patients with adenocarcinoma treated with carboplatin/taxol prior to surgery (+Chemo) . Shown are expression profile comparisons for A , TARS, B , VEGF, C , SARS and D , YARS. Differences between groups was significant for TARS and VEGF as determined by ANOVA Kruskal Wallis analysis, *p < 0.0001 compared with benign, #p < 0.001 compared with carcinoma.

Article Snippet: Cell lysates (5%) and corresponding concentrated cell media (25%) were separated by SDS-PAGE and analyzed by Western blot for presence of TARS (1:1000 rabbit-anti-ThrRS, Santa Cruz Biotechnology SC-98543) as described in [ ]. β-tubulin was detected as a loading control for the lysates and to confirm that TARS found in the media was not a result of cell lysis.

Techniques: Adjuvant, Microarray, Expressing

Journal: BMC Cancer

Article Title: Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer

doi: 10.1186/1471-2407-14-620

Figure Lengend Snippet: TARS expression increases with stage of ovarian cancer. A . Tissues were stained for TARS by immunohistochemistry and counter-stained with Mayers’ hematoxylin. Images were scored blindly by 2 independent investigators and tumor identification was later confirmed by a pathologist (SLM). Shown are 10x images from (1) normal ovary and examples of TARS score increasing with extent of disease (2–4), Bar = 100 μm. B . Graph representing average scores where high stage is ≥ stage 3; statistical significance determined by one-way ANOVA. For more detailed statistics, see Table for Pearson’s Correlations. C . Shown is a graph of individual patient TARS tumor scores grouped according to FIGO stage of disease (i.e. Stage 3.75 = FIGO 3C).

Article Snippet: Cell lysates (5%) and corresponding concentrated cell media (25%) were separated by SDS-PAGE and analyzed by Western blot for presence of TARS (1:1000 rabbit-anti-ThrRS, Santa Cruz Biotechnology SC-98543) as described in [ ]. β-tubulin was detected as a loading control for the lysates and to confirm that TARS found in the media was not a result of cell lysis.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: BMC Cancer

Article Title: Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer

doi: 10.1186/1471-2407-14-620

Figure Lengend Snippet: Pearson Correlation Matrix for TARS expression in ovarian cancer patient samples

Article Snippet: Cell lysates (5%) and corresponding concentrated cell media (25%) were separated by SDS-PAGE and analyzed by Western blot for presence of TARS (1:1000 rabbit-anti-ThrRS, Santa Cruz Biotechnology SC-98543) as described in [ ]. β-tubulin was detected as a loading control for the lysates and to confirm that TARS found in the media was not a result of cell lysis.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer

doi: 10.1186/1471-2407-14-620

Figure Lengend Snippet: TARS is secreted by SK - OV - 3 ovarian cancer cells. (A) SK-OV-3 cells were treated with TNF-α (50 ng/ml) or exposed to 2% O 2 for 24 h where indicated. Media was concentrated 20-fold to accommodate 25% onto the gel and compared to 5% of the cell lysate. Shown is a representative Western blot probed for TARS and β-tubulin, n = 4. (B) Cells were treated as in (A) . After 24 h the level of TARS in the supernatant was determined by ELISA, *p < 0.05, n = 3. Cell membrane integrity was confirmed using the lactate dehydrogenase assay CytoTox-ONE™ and by lack of β-tubulin in media samples. (C) Relative mRNA levels for TARS, VEGF and IL-1β were determined by RT-qPCR following treatment as in (A) ; *p < 0.001, **p < 0.0001, n = 4.

Article Snippet: Cell lysates (5%) and corresponding concentrated cell media (25%) were separated by SDS-PAGE and analyzed by Western blot for presence of TARS (1:1000 rabbit-anti-ThrRS, Santa Cruz Biotechnology SC-98543) as described in [ ]. β-tubulin was detected as a loading control for the lysates and to confirm that TARS found in the media was not a result of cell lysis.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Membrane, Lactate Dehydrogenase Assay, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer

doi: 10.1186/1471-2407-14-620

Figure Lengend Snippet: Correlation of TARS levels detected in serum with TARS expression in ovarian cancer. Graphs represent scatter dot plots (mean ± SD) of patient data for TARS serum levels against (A) Stage of cancer and (B) TARS tumor tissue score. The correlation (r-value) and p-values were calculated by a Pearson correlation matrix (see Table ).

Article Snippet: Cell lysates (5%) and corresponding concentrated cell media (25%) were separated by SDS-PAGE and analyzed by Western blot for presence of TARS (1:1000 rabbit-anti-ThrRS, Santa Cruz Biotechnology SC-98543) as described in [ ]. β-tubulin was detected as a loading control for the lysates and to confirm that TARS found in the media was not a result of cell lysis.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer

doi: 10.1186/1471-2407-14-620

Figure Lengend Snippet: Hazard ratios for the association of TARS with mortality using univariate Cox proportional hazard models

Article Snippet: Cell lysates (5%) and corresponding concentrated cell media (25%) were separated by SDS-PAGE and analyzed by Western blot for presence of TARS (1:1000 rabbit-anti-ThrRS, Santa Cruz Biotechnology SC-98543) as described in [ ]. β-tubulin was detected as a loading control for the lysates and to confirm that TARS found in the media was not a result of cell lysis.

Techniques:

Journal: BMC Cancer

Article Title: Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer

doi: 10.1186/1471-2407-14-620

Figure Lengend Snippet: Multivariate Cox proportional hazard model for TARS tumor score

Article Snippet: Cell lysates (5%) and corresponding concentrated cell media (25%) were separated by SDS-PAGE and analyzed by Western blot for presence of TARS (1:1000 rabbit-anti-ThrRS, Santa Cruz Biotechnology SC-98543) as described in [ ]. β-tubulin was detected as a loading control for the lysates and to confirm that TARS found in the media was not a result of cell lysis.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: Loss of MTX2 causes mitochondrial dysfunction, podocyte injury, nephrotic proteinuria and glomerulopathy in mice and patients

doi: 10.7150/ijbs.89916

Figure Lengend Snippet: Clinical manifestations and mutations of gene MTX2 in probands. (A) Photograph of patient 1. The boy showed specific facial features (sparse hair; frontal bossing; exophthalmos; prominent ear; thick lip; crowding teeth; micrognathia). (B) Renal ultrasound of patient 1. (C) Cranial CT in patient 1. (D) Echocardiography of patient 1. (E) Representative images of PASM staining in kidney of patient 2. yellow arrows, “pseudo double track”, red arrows, thickening of basement membrane, scale bar: 25 μm. (F) Masson staining in kidney of patient 2. red arrows, deposition of fuchsinophilic protein in mesangial area and basement membrane, scale bar: 25 μm. (G) Transmission EM images in kidney of patient 2. red arrows, electronic dense deposits in subendothelial, mesangial areas and subepithelial, scale bar: 5 μm. (H) MTX2 mutations in families. The compound heterozygous mutation including c.378+1 (IVS6) G>A mutation and c.95 (exon3) delA mutation in patient 1 and the c.500 (exon8) _c.501 (exon8) insT homozygous mutation in patient 2 was identified by WES. Circles indicate females; squares indicate males; solid indicate patients; arrows indicate patients.

Article Snippet: Antibodies used in this work were as follows: MTX2 (11610-1-AP, Proteintech) diluted at 1:250 for immunohistochemistry (IHC) and 1:600 for western blotting (WB); MTX2 (sc-514231, Santa Cruz) diluted at 1:250 for immunofluorescence (IF); synaptopodin (21064-1-AP, Proteintech) diluted at 1:400 for IF; Alexa Fluor 594 donkey anti-rabbit IgG (H+L) secondary antibody (A21207; Life Technologies) diluted at 1:500 for IF; Alexa Fluor 488 Goat anti-mouse IgG, IgM (H+L) secondary antibody (A-10680, Invitrogen) diluted at 1:10000 for IF; WT1 (DF6331, Affinity Biosciences) diluted at 1:250 for IF; ND2 (A17968, ABclonal) diluted at 1:1000 for WB; ND4 (A9941, ABclonal) diluted at 1:1000 for WB; ND5 (A17972, ABclonal) diluted at 1:1000 for WB; CYTB (A17966, ABclonal) diluted at 1:1000 for WB; MTCO2 (55070-1-AP, Proteintech) diluted at 1:2000 for WB; NDUFA9 (A3196, ABclonal) diluted at 1:1000 for WB; NDUFS1 (12444-1-AP, Proteintech) diluted at 1:5000 for WB; SDHB (10620-1-AP, Proteintech) diluted at 1: 20000 for WB; UQCRC1 (21705-1-AP, Proteintech) diluted at 1:4000 for WB; ATPB (17247-1-AP, Proteintech) diluted at 1:3000 for WB; TOM20 (A19403, ABclonal) diluted at 1:5000 for WB; MFN2 (12186-1-AP, Proteintech) diluted at 1: 5000 for WB; OPA1 (27733-1-AP, Proteintech) diluted at 1:2000 for WB; DRP1 (12957-1-AP, Proteintech) diluted at 1:5000 for WB; FIS1 (10956-1-AP, Proteintech) diluted at 1:1500 for WB; LC3B (ab192890, Abcam) diluted at 1:2000 for WB; Metaxin 1 (sc-135989, Santa Cruz) diluted at 1:500 for WB; VDAC1/2 (10866-1-AP, Proteintech) diluted at 1:1500 for WB; TOMM40 (18409-1-AP, Proteintech) diluted at 1:8000 for WB; Sam50 (28679-1-AP, Proteintech) diluted at 1:8000 for WB; Mitofilin (10179-1-AP, Proteintech) diluted at 1:1000 for WB; CHCHD3 (25625-1-AP, Proteintech) diluted at 1:8000 for WB.

Techniques: Staining, Membrane, Transmission Assay, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: Loss of MTX2 causes mitochondrial dysfunction, podocyte injury, nephrotic proteinuria and glomerulopathy in mice and patients

doi: 10.7150/ijbs.89916

Figure Lengend Snippet: Renal phenotype in Pod-Mtx2-KO mice. (A) Western blots and quantification analysis, showing reduced MTX2 expression in primary glomerular cells from Pod-Mtx2-KO mice compared with control mice (n = 3). (B) The summary data on body weights of mice (n = 10). (C) The summary data on kidney weights of mice (n = 10). (D) Representative images of kidney from Pod-Mtx2-KO and control mice at 3, 6, and 9 mo of age. (E) 24 h urinary albumin quantification of Pod-Mtx2-KO and control mice at 8, 12, 16 weeks of age (n = 10). (F) Coomassie blue staining of urine from Pod-Mtx2-KO and control mice at 12 weeks of age as well as standard BSA with different concentrations. (G) Representative images showing F-actin cytoskeleton stained with FITC labeled phalloidin in primary podocytes isolated from Pod-Mtx2-KO and control mice at 8 weeks of age, scale bars: 50 μm. WT1, a specific marker protein of podocytes to identify isolated primary glomerular podocytes. Data was shown as the mean ± SD of triplicates at least. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: Antibodies used in this work were as follows: MTX2 (11610-1-AP, Proteintech) diluted at 1:250 for immunohistochemistry (IHC) and 1:600 for western blotting (WB); MTX2 (sc-514231, Santa Cruz) diluted at 1:250 for immunofluorescence (IF); synaptopodin (21064-1-AP, Proteintech) diluted at 1:400 for IF; Alexa Fluor 594 donkey anti-rabbit IgG (H+L) secondary antibody (A21207; Life Technologies) diluted at 1:500 for IF; Alexa Fluor 488 Goat anti-mouse IgG, IgM (H+L) secondary antibody (A-10680, Invitrogen) diluted at 1:10000 for IF; WT1 (DF6331, Affinity Biosciences) diluted at 1:250 for IF; ND2 (A17968, ABclonal) diluted at 1:1000 for WB; ND4 (A9941, ABclonal) diluted at 1:1000 for WB; ND5 (A17972, ABclonal) diluted at 1:1000 for WB; CYTB (A17966, ABclonal) diluted at 1:1000 for WB; MTCO2 (55070-1-AP, Proteintech) diluted at 1:2000 for WB; NDUFA9 (A3196, ABclonal) diluted at 1:1000 for WB; NDUFS1 (12444-1-AP, Proteintech) diluted at 1:5000 for WB; SDHB (10620-1-AP, Proteintech) diluted at 1: 20000 for WB; UQCRC1 (21705-1-AP, Proteintech) diluted at 1:4000 for WB; ATPB (17247-1-AP, Proteintech) diluted at 1:3000 for WB; TOM20 (A19403, ABclonal) diluted at 1:5000 for WB; MFN2 (12186-1-AP, Proteintech) diluted at 1: 5000 for WB; OPA1 (27733-1-AP, Proteintech) diluted at 1:2000 for WB; DRP1 (12957-1-AP, Proteintech) diluted at 1:5000 for WB; FIS1 (10956-1-AP, Proteintech) diluted at 1:1500 for WB; LC3B (ab192890, Abcam) diluted at 1:2000 for WB; Metaxin 1 (sc-135989, Santa Cruz) diluted at 1:500 for WB; VDAC1/2 (10866-1-AP, Proteintech) diluted at 1:1500 for WB; TOMM40 (18409-1-AP, Proteintech) diluted at 1:8000 for WB; Sam50 (28679-1-AP, Proteintech) diluted at 1:8000 for WB; Mitofilin (10179-1-AP, Proteintech) diluted at 1:1000 for WB; CHCHD3 (25625-1-AP, Proteintech) diluted at 1:8000 for WB.

Techniques: Western Blot, Expressing, Control, Staining, Labeling, Isolation, Marker

Journal: International Journal of Biological Sciences

Article Title: Loss of MTX2 causes mitochondrial dysfunction, podocyte injury, nephrotic proteinuria and glomerulopathy in mice and patients

doi: 10.7150/ijbs.89916

Figure Lengend Snippet: Renal histology and ultrastructural morphology in Pod-Mtx2-KO mice. (A) HE staining of glomerular mesangial hyperplasia in different degrees in Pod-Mtx2-KO mice kidneys during the 40 week follow-up (n = 6), yellow arrows, mesangial cells, scale bar: 50 μm. (B) Images of HE staining demonstrating no significant difference in renal tubules between Pod-Mtx2-KO and control mice during the 40 week follow-up (n = 6), scale bar: 100 μm. (C) Transmission EM images, showing fusion of podocyte foot processes, abnormal basement membrane and mesangial matrix proliferation in glomerulus of Pod-Mtx2-KO mice at different ages (n = 6), scale bar: 5 μm. (D) Images of scanning EM showing abnormal podocyte foot process architecture in Pod-Mtx2-KO mice at different ages (n = 6), scale bar: 2 μm. (E) Images of transmission EM illustrating mitochondrial structural abnormalities and increased autophagosomes in podocytes of Pod-Mtx2-KO mice during the 40 week follow-up (n = 6), yellow arrows, abnormal structure of mitochondria; red asterisk, autophagosomes, scale bar: 250 nm.

Article Snippet: Antibodies used in this work were as follows: MTX2 (11610-1-AP, Proteintech) diluted at 1:250 for immunohistochemistry (IHC) and 1:600 for western blotting (WB); MTX2 (sc-514231, Santa Cruz) diluted at 1:250 for immunofluorescence (IF); synaptopodin (21064-1-AP, Proteintech) diluted at 1:400 for IF; Alexa Fluor 594 donkey anti-rabbit IgG (H+L) secondary antibody (A21207; Life Technologies) diluted at 1:500 for IF; Alexa Fluor 488 Goat anti-mouse IgG, IgM (H+L) secondary antibody (A-10680, Invitrogen) diluted at 1:10000 for IF; WT1 (DF6331, Affinity Biosciences) diluted at 1:250 for IF; ND2 (A17968, ABclonal) diluted at 1:1000 for WB; ND4 (A9941, ABclonal) diluted at 1:1000 for WB; ND5 (A17972, ABclonal) diluted at 1:1000 for WB; CYTB (A17966, ABclonal) diluted at 1:1000 for WB; MTCO2 (55070-1-AP, Proteintech) diluted at 1:2000 for WB; NDUFA9 (A3196, ABclonal) diluted at 1:1000 for WB; NDUFS1 (12444-1-AP, Proteintech) diluted at 1:5000 for WB; SDHB (10620-1-AP, Proteintech) diluted at 1: 20000 for WB; UQCRC1 (21705-1-AP, Proteintech) diluted at 1:4000 for WB; ATPB (17247-1-AP, Proteintech) diluted at 1:3000 for WB; TOM20 (A19403, ABclonal) diluted at 1:5000 for WB; MFN2 (12186-1-AP, Proteintech) diluted at 1: 5000 for WB; OPA1 (27733-1-AP, Proteintech) diluted at 1:2000 for WB; DRP1 (12957-1-AP, Proteintech) diluted at 1:5000 for WB; FIS1 (10956-1-AP, Proteintech) diluted at 1:1500 for WB; LC3B (ab192890, Abcam) diluted at 1:2000 for WB; Metaxin 1 (sc-135989, Santa Cruz) diluted at 1:500 for WB; VDAC1/2 (10866-1-AP, Proteintech) diluted at 1:1500 for WB; TOMM40 (18409-1-AP, Proteintech) diluted at 1:8000 for WB; Sam50 (28679-1-AP, Proteintech) diluted at 1:8000 for WB; Mitofilin (10179-1-AP, Proteintech) diluted at 1:1000 for WB; CHCHD3 (25625-1-AP, Proteintech) diluted at 1:8000 for WB.

Techniques: Staining, Control, Transmission Assay, Membrane

Journal: International Journal of Biological Sciences

Article Title: Loss of MTX2 causes mitochondrial dysfunction, podocyte injury, nephrotic proteinuria and glomerulopathy in mice and patients

doi: 10.7150/ijbs.89916

Figure Lengend Snippet: Podocyte function in MPC5 with or without MTX2 and apoptosis protein microarray assay. (A) Quantification of adherent podocytes after podocytes were seeded on plates coated with collagen for 1 h. (B) Representative images and quantification of migrating podocytes after migration through transwell chamber membranes for 24 h, scale bar: 250 μm. (C) Quantification of the mean fluorescence intensity of FITC-transferrin engulfed by podocytes for 5 h. (D) Quantification of attached podocytes with or without puromycin aminonucleoside (PAN) treatment. (E) F-actin cytoskeleton stained with FITC labeled phalloidin in podocytes, scale bar: 50 μm. (F) Podocyte apoptosis measured by propidium iodide (PI) and FITC-conjugated Annexin V. (G) Representative images of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis by mouse apoptosis protein microarray performed on MTX2-KD and control podocytes. (H) Representative heatmap of mouse apoptosis associated proteins by mouse apoptosis protein microarray. Data was shown as the mean ± SD of triplicates at least. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: Antibodies used in this work were as follows: MTX2 (11610-1-AP, Proteintech) diluted at 1:250 for immunohistochemistry (IHC) and 1:600 for western blotting (WB); MTX2 (sc-514231, Santa Cruz) diluted at 1:250 for immunofluorescence (IF); synaptopodin (21064-1-AP, Proteintech) diluted at 1:400 for IF; Alexa Fluor 594 donkey anti-rabbit IgG (H+L) secondary antibody (A21207; Life Technologies) diluted at 1:500 for IF; Alexa Fluor 488 Goat anti-mouse IgG, IgM (H+L) secondary antibody (A-10680, Invitrogen) diluted at 1:10000 for IF; WT1 (DF6331, Affinity Biosciences) diluted at 1:250 for IF; ND2 (A17968, ABclonal) diluted at 1:1000 for WB; ND4 (A9941, ABclonal) diluted at 1:1000 for WB; ND5 (A17972, ABclonal) diluted at 1:1000 for WB; CYTB (A17966, ABclonal) diluted at 1:1000 for WB; MTCO2 (55070-1-AP, Proteintech) diluted at 1:2000 for WB; NDUFA9 (A3196, ABclonal) diluted at 1:1000 for WB; NDUFS1 (12444-1-AP, Proteintech) diluted at 1:5000 for WB; SDHB (10620-1-AP, Proteintech) diluted at 1: 20000 for WB; UQCRC1 (21705-1-AP, Proteintech) diluted at 1:4000 for WB; ATPB (17247-1-AP, Proteintech) diluted at 1:3000 for WB; TOM20 (A19403, ABclonal) diluted at 1:5000 for WB; MFN2 (12186-1-AP, Proteintech) diluted at 1: 5000 for WB; OPA1 (27733-1-AP, Proteintech) diluted at 1:2000 for WB; DRP1 (12957-1-AP, Proteintech) diluted at 1:5000 for WB; FIS1 (10956-1-AP, Proteintech) diluted at 1:1500 for WB; LC3B (ab192890, Abcam) diluted at 1:2000 for WB; Metaxin 1 (sc-135989, Santa Cruz) diluted at 1:500 for WB; VDAC1/2 (10866-1-AP, Proteintech) diluted at 1:1500 for WB; TOMM40 (18409-1-AP, Proteintech) diluted at 1:8000 for WB; Sam50 (28679-1-AP, Proteintech) diluted at 1:8000 for WB; Mitofilin (10179-1-AP, Proteintech) diluted at 1:1000 for WB; CHCHD3 (25625-1-AP, Proteintech) diluted at 1:8000 for WB.

Techniques: Microarray, Migration, Fluorescence, Staining, Labeling, Control

Journal: International Journal of Biological Sciences

Article Title: Loss of MTX2 causes mitochondrial dysfunction, podocyte injury, nephrotic proteinuria and glomerulopathy in mice and patients

doi: 10.7150/ijbs.89916

Figure Lengend Snippet: Mitochondrial function in MPC5 with or without MTX2. (A) Western blots and quantifications analysis of some subunits in respiratory chain complex. TOM20 as the loading control. (B) Quantifications of enzyme activities of oxidative respiratory chains (complex I, III and IV) in MTX2-KD and control podocytes. (C) Quantifications of whole cell ATP production in MPC5 with MTX2 knockdown. (D) Measurement of MMP using the fluorescence probe JC-1 assay kit in MTX2-KD and control podocytes. (E) The OCR curves of podocytes with and without MTX2, stimulated with different inhibitors (oligomycin (1.5 μM), FCCP (2 μM), Retenone /Antimycin (0.5 μM)) at specific time points and quantification of the OCRs. (F) The OCR curves of podocytes with MTX2 overexpressing, compared with the control. (G) Mitochondrial ROS assayed by flow cytometry using the fluorescent probe DCFH-DA in MTX2-KD and control podocytes. (H) Mitochondrial ROS measurement with MTX2 overexpression, compared to the controls. Data was shown as the mean ± SD of triplicates at least. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: Antibodies used in this work were as follows: MTX2 (11610-1-AP, Proteintech) diluted at 1:250 for immunohistochemistry (IHC) and 1:600 for western blotting (WB); MTX2 (sc-514231, Santa Cruz) diluted at 1:250 for immunofluorescence (IF); synaptopodin (21064-1-AP, Proteintech) diluted at 1:400 for IF; Alexa Fluor 594 donkey anti-rabbit IgG (H+L) secondary antibody (A21207; Life Technologies) diluted at 1:500 for IF; Alexa Fluor 488 Goat anti-mouse IgG, IgM (H+L) secondary antibody (A-10680, Invitrogen) diluted at 1:10000 for IF; WT1 (DF6331, Affinity Biosciences) diluted at 1:250 for IF; ND2 (A17968, ABclonal) diluted at 1:1000 for WB; ND4 (A9941, ABclonal) diluted at 1:1000 for WB; ND5 (A17972, ABclonal) diluted at 1:1000 for WB; CYTB (A17966, ABclonal) diluted at 1:1000 for WB; MTCO2 (55070-1-AP, Proteintech) diluted at 1:2000 for WB; NDUFA9 (A3196, ABclonal) diluted at 1:1000 for WB; NDUFS1 (12444-1-AP, Proteintech) diluted at 1:5000 for WB; SDHB (10620-1-AP, Proteintech) diluted at 1: 20000 for WB; UQCRC1 (21705-1-AP, Proteintech) diluted at 1:4000 for WB; ATPB (17247-1-AP, Proteintech) diluted at 1:3000 for WB; TOM20 (A19403, ABclonal) diluted at 1:5000 for WB; MFN2 (12186-1-AP, Proteintech) diluted at 1: 5000 for WB; OPA1 (27733-1-AP, Proteintech) diluted at 1:2000 for WB; DRP1 (12957-1-AP, Proteintech) diluted at 1:5000 for WB; FIS1 (10956-1-AP, Proteintech) diluted at 1:1500 for WB; LC3B (ab192890, Abcam) diluted at 1:2000 for WB; Metaxin 1 (sc-135989, Santa Cruz) diluted at 1:500 for WB; VDAC1/2 (10866-1-AP, Proteintech) diluted at 1:1500 for WB; TOMM40 (18409-1-AP, Proteintech) diluted at 1:8000 for WB; Sam50 (28679-1-AP, Proteintech) diluted at 1:8000 for WB; Mitofilin (10179-1-AP, Proteintech) diluted at 1:1000 for WB; CHCHD3 (25625-1-AP, Proteintech) diluted at 1:8000 for WB.

Techniques: Western Blot, Control, Knockdown, Fluorescence, Flow Cytometry, Over Expression

Journal: International Journal of Biological Sciences

Article Title: Loss of MTX2 causes mitochondrial dysfunction, podocyte injury, nephrotic proteinuria and glomerulopathy in mice and patients

doi: 10.7150/ijbs.89916

Figure Lengend Snippet: Alterations in mitochondrial dynamics, structure, membrane proteins and the Sam50-CHCHD3-Mitofilin axis. (A) Western blots and quantification analysis of mitochondrial membrane proteins (MTX1, VDAC, TOM40) in MTX2-KD and control podocytes. (B) Western blots and quantification analysis of the Sam50-CHCHD3-Mitofilin axis in MTX2-KD and control podocytes. (C) Images of mitochondrial ultrastructure by transmission EM in MTX2-KD podocytes. yellow arrows, abnormal structure of mitochondria; red asterisk, autophagosomes, scale bars: 2 μm. (D) Western blots assay in MTX2-KD podocytes compared to controls, observing decreased levels of mitochondrial fusion proteins MFN2 and OPA1 (including OPA1-L and OPA1-S) and increased levels of mitochondrial fission proteins DRP1 and FIS1. (E) Western blots and quantification analysis showing increased LC3-II/I ratio in MTX2-KD podocytes. Data was shown as the mean ± SD of triplicates at least. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: Antibodies used in this work were as follows: MTX2 (11610-1-AP, Proteintech) diluted at 1:250 for immunohistochemistry (IHC) and 1:600 for western blotting (WB); MTX2 (sc-514231, Santa Cruz) diluted at 1:250 for immunofluorescence (IF); synaptopodin (21064-1-AP, Proteintech) diluted at 1:400 for IF; Alexa Fluor 594 donkey anti-rabbit IgG (H+L) secondary antibody (A21207; Life Technologies) diluted at 1:500 for IF; Alexa Fluor 488 Goat anti-mouse IgG, IgM (H+L) secondary antibody (A-10680, Invitrogen) diluted at 1:10000 for IF; WT1 (DF6331, Affinity Biosciences) diluted at 1:250 for IF; ND2 (A17968, ABclonal) diluted at 1:1000 for WB; ND4 (A9941, ABclonal) diluted at 1:1000 for WB; ND5 (A17972, ABclonal) diluted at 1:1000 for WB; CYTB (A17966, ABclonal) diluted at 1:1000 for WB; MTCO2 (55070-1-AP, Proteintech) diluted at 1:2000 for WB; NDUFA9 (A3196, ABclonal) diluted at 1:1000 for WB; NDUFS1 (12444-1-AP, Proteintech) diluted at 1:5000 for WB; SDHB (10620-1-AP, Proteintech) diluted at 1: 20000 for WB; UQCRC1 (21705-1-AP, Proteintech) diluted at 1:4000 for WB; ATPB (17247-1-AP, Proteintech) diluted at 1:3000 for WB; TOM20 (A19403, ABclonal) diluted at 1:5000 for WB; MFN2 (12186-1-AP, Proteintech) diluted at 1: 5000 for WB; OPA1 (27733-1-AP, Proteintech) diluted at 1:2000 for WB; DRP1 (12957-1-AP, Proteintech) diluted at 1:5000 for WB; FIS1 (10956-1-AP, Proteintech) diluted at 1:1500 for WB; LC3B (ab192890, Abcam) diluted at 1:2000 for WB; Metaxin 1 (sc-135989, Santa Cruz) diluted at 1:500 for WB; VDAC1/2 (10866-1-AP, Proteintech) diluted at 1:1500 for WB; TOMM40 (18409-1-AP, Proteintech) diluted at 1:8000 for WB; Sam50 (28679-1-AP, Proteintech) diluted at 1:8000 for WB; Mitofilin (10179-1-AP, Proteintech) diluted at 1:1000 for WB; CHCHD3 (25625-1-AP, Proteintech) diluted at 1:8000 for WB.

Techniques: Membrane, Western Blot, Control, Transmission Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Gasdermin D promotes development of intestinal tumors through regulating IL-1β release and gut microbiota composition

doi: 10.1186/s12964-024-01890-6

Figure Lengend Snippet: GSDMD is upregulated in intestinal cancer. ( A ) IHC analysis of GSDMD protein level in a human tissue microarray, including 80 colorectal cancer and matched adjacent tumor specimens. ( B ) IHC analysis of GSDMD protein level in four colorectal cancer tissue and matched adjacent cancer tissue from ( A ) (200× magnification). ( C - E ) GSDMD expression score in cytoplasm ( C ), nuclear ( D ), and cytoplasm + nuclear ( E ) as calculated by multiplying the intensity and positive percentage scores according to ( A ). ( F ) Western blot analysis of GSDMD in in small intestine tumor tissue from Apc min/+ mice ( n = 5) and normal tissue from WT mice ( n = 3). Data are representative of at least three independent experiments (mean ± SEM). *** p < 0.001 by Student’s t test. FL means full length

Article Snippet: Anti-GSDMD antibody (Proteintech) was used to evaluate GSDMD expression in intestinal cancer via immunohistochemistry (IHC).

Techniques: Microarray, Expressing, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Gasdermin D promotes development of intestinal tumors through regulating IL-1β release and gut microbiota composition

doi: 10.1186/s12964-024-01890-6

Figure Lengend Snippet: Deficiency of GSDMD suppresses spontaneous intestinal cancer development. ( A ) Macroscopic view of representative small intestine from 20-week old Apc min/+ and Apc min/+ Gsdmd −/− mice. ( B ) Hematoxylin and eosin (H&E) staining of the representative intestinal tumor from the Apc min/+ and Apc min/+ Gsdmd −/− mice as in ( A ) (100× magnification). ( C - F ) Tumor number ( C and E ) and tumor load ( D and F ) from the small intestines or colons of 20-week-old Apc min/+ ( n = 7) and Apc min/+ Gsdmd −/− ( n = 7) mice. ( G ) Histogram showing the size distribution of tumors from the small intestines of 20-week-old Apc min/+ ( n = 7) and Apc min/+ Gsdmd −/− ( n = 7) mice. ( H - J ) Hematocrit ( H ), thymus weight ( I ) and spleen weight ( J ) of 20-week-old Apc min/+ ( n = 6–7) and Apc min/+ Gsdmd −/− ( n = 6–7) mice. Data are representative of at least three independent experiments (mean ± SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 by Student’s t test

Article Snippet: Anti-GSDMD antibody (Proteintech) was used to evaluate GSDMD expression in intestinal cancer via immunohistochemistry (IHC).

Techniques: Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Gasdermin D promotes development of intestinal tumors through regulating IL-1β release and gut microbiota composition

doi: 10.1186/s12964-024-01890-6

Figure Lengend Snippet: Exogenous IL-1β promotes intestinal tumor development in Apc min/+ Gsdmd −/− mice. ( A ) 8-week-old Apc min/+ Gsdmd −/− mice simultaneously received injection of IL-1β or PBS twice a week, while age and sex-matched Apc min/+ mice were injected with PBS ( n = 5–6/group). H&E staining of the representative intestinal tumor from the 20-week old above mice (100× magnification). Tumors are circled with dashed lines. ( B - E ) Tumor number ( B and D ) and tumor load ( C and E ) from the small intestines or colons of 20-week-old above mice as in ( A ). ( F - G ) Spleen weight ( F ), and hematocrit ( G ) of 20-week-old above mice as in ( A ). ( H ) Histogram showing the size distribution of tumors from the small intestines of 20-week-old above mice as in ( A ). Data are representative of at least three independent experiments (mean ± SEM). ** p < 0.01 by Student’s t test. N.S. means no significance

Article Snippet: Anti-GSDMD antibody (Proteintech) was used to evaluate GSDMD expression in intestinal cancer via immunohistochemistry (IHC).

Techniques: Injection, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Gasdermin D promotes development of intestinal tumors through regulating IL-1β release and gut microbiota composition

doi: 10.1186/s12964-024-01890-6

Figure Lengend Snippet: Exogenous Kyn promotes intestinal tumor development in Apc min/+ Gsdmd −/− mice. ( A ) ELISA analysis of Kyn and Trp from colon of Apc min/+ ( n = 6) and Apc min/+ Gsdmd −/− ( n = 6) mice. ( B ) Kyn/Trp was determined as in ( A ). ( C ) 8-week-old Apc min/+ Gsdmd −/− mice simultaneously received injection of Kyn or PBS once a week, while age and sex-matched Apc min/+ mice were injected with PBS ( n = 5/group). H&E staining of the representative intestinal tumor from the 20-week old above mice (100× magnification). ( D - G ) Tumor number ( D and F ) and tumor load ( E and G ) from the small intestines or colons of 20-week-old above mice as in ( C ). ( H - I ) Spleen weight ( H ), and hematocrit ( I ) of 20-week-old above mice as in ( C ). ( J ) Histogram showing the size distribution of tumors from the small intestines of 20-week-old above mice as in ( C ). Data are representative of at least three independent experiments (mean ± SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 by Student’s t test. N.S. means no significance

Article Snippet: Anti-GSDMD antibody (Proteintech) was used to evaluate GSDMD expression in intestinal cancer via immunohistochemistry (IHC).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Gasdermin D promotes development of intestinal tumors through regulating IL-1β release and gut microbiota composition

doi: 10.1186/s12964-024-01890-6

Figure Lengend Snippet: Exogenous Kyn increases Treg cell number in Apc min/+ Gsdmd −/− mice. ( A ) 8-week-old Apc min/+ Gsdmd −/− mice simultaneously received injection of Kyn or PBS once a week, while age and sex-matched Apc min/+ mice were injected with PBS ( n = 4/group). Flow cytometry analysis of CD4 + and CD8 + T cell number from intestinal tumor of the 20-week old above mice. ( B ) Total leukocytes were determined by CD45 + cell. CD4 + T cells % of total leukocyte and CD8 + T cells % of total leukocyte were determined as in ( A ). ( C ) Flow cytometry analysis of Treg (CD4 + Foxp3 + ) cell number from intestinal tumor of the 20-week old mice as in ( A ). ( D ) Treg % of CD4 + cells was determined as in ( C ). Data are representative of at least three independent experiments (mean ± SEM). ** p < 0.01, *** p < 0.001 by Student’s t test. N.S. means no significance

Article Snippet: Anti-GSDMD antibody (Proteintech) was used to evaluate GSDMD expression in intestinal cancer via immunohistochemistry (IHC).

Techniques: Injection, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Comprehensive analyses of telomerase component DKC1 and its association with clinical, molecular and immune landscapes in uterine corpus endometrial carcinoma

doi: 10.3389/fcell.2025.1592135

Figure Lengend Snippet: DKC1 expression is upregulated in UCEC tumors (A) The flow chart of the study (B) The levels of DKC1 mRNA [log2 (TPM+1)] were evaluated and compared between 545 tumors and 35 non-tumorous endometrial tissues in the TCGA UCEC cohort (C) The DKC1 protein levels (Z-value) were evaluated and compared between 100 tumors and 31 non-tumorous endometrial tissues in the CPTCA UCEC cohort (D) The significantly positive correlation between mRNA and protein levels of DKC1 (Z-value) based on (B) and (C) results (E–G) The upregulation of DKC1 expression in UCEC tumors from the Qilu cohort, as determined using immunohistochemistry (IHC). The representative IHC images in (E) showed stronger DKC1 staining in tumors than in adjacent normal glands. Magnifications: ×40 (F) Comparison of IHC scores between tumors and adjacent normal glands in 12 paired samples (G) Comparison of IHC scores in all 30 tumors with 12 normal gland-containing samples.

Article Snippet: Slides were blocked using 10% goat serum and incubated with a DKC1 polyclonal antibody (Cat 25420-1-AP, Proteintech, Rosemont, IL) for 2 h at room temperature.

Techniques: Expressing, Immunohistochemistry, Staining, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Comprehensive analyses of telomerase component DKC1 and its association with clinical, molecular and immune landscapes in uterine corpus endometrial carcinoma

doi: 10.3389/fcell.2025.1592135

Figure Lengend Snippet: The positive correlation between higher DKC1 expression and TERC, telomerase activity and aggressive UCEC tumors (A–F) RNA levels were assessed using log2 (TPM+1). The TCGA cohort of UCEC was analyzed (A) Upregulation of TERC expression in UCEC tumors (B) The positive correlation between DKC1 and TERC expression (C, D) The positive correlation between DKC1 and telomerase activity. Telomerase activity levels were calculated using the telomerase score (Ref. 14) and EXTEND (Ref. 41) algorithms, respectively (E) Significantly higher DKC1 expression in serous and mixed types of UCECs (F) The association between higher DKC1 expression and higher risk of recurrence (G) The association between higher DKC1 expression and higher frequency of metastasis. The GSE120490 UCEC cohort with 145 UCEC patients (100 without and 45 with metastasis) were analyzed (H) Significantly higher DKC1 expression (microarray data) in late-stage UCEC tumors from the GSE23518 cohort (with 10 early and 10 late-stage UCECs).

Article Snippet: Slides were blocked using 10% goat serum and incubated with a DKC1 polyclonal antibody (Cat 25420-1-AP, Proteintech, Rosemont, IL) for 2 h at room temperature.

Techniques: Expressing, Activity Assay, Microarray

Journal: Frontiers in Cell and Developmental Biology

Article Title: Comprehensive analyses of telomerase component DKC1 and its association with clinical, molecular and immune landscapes in uterine corpus endometrial carcinoma

doi: 10.3389/fcell.2025.1592135

Figure Lengend Snippet: Higher DKC1 expression predicts UCEC patient survival independently. Patients in the TCGA UCEC cohort were categorized into low and high groups based DKC1 mRNA levels in their tumors (median value as the cutoff) (A, B) Association between DKC1 expression and overall and progression-free survival (OS and PFS) (C, D) Univariate and multivariate COX regression analyses of DKC1 effect on patient OS (C) Univariate and (D) Multivariate (E, F) Univariate and multivariate COX regression analyses of DKC1 effect on patient PFS (E) Univariate and (F) Multivariate (G–I) Nomogram for prediction of UCEC PFS. A total of 349 patients were analyzed by including DKC1 (high vs. low), stage (I/II vs. III/IV) and age (<60 vs. ≥60) (H) The accuracy of the nomogram to predict PFS (Prediction curve vs. observed scenario) (I) The ROC prediction of PFS. ROC showed AUC values 0.67, 0.73 and 0.71 at 1, 3 and 5 years PFS, respectively. RNA levels were calculated using log2 (TPM+1).

Article Snippet: Slides were blocked using 10% goat serum and incubated with a DKC1 polyclonal antibody (Cat 25420-1-AP, Proteintech, Rosemont, IL) for 2 h at room temperature.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Comprehensive analyses of telomerase component DKC1 and its association with clinical, molecular and immune landscapes in uterine corpus endometrial carcinoma

doi: 10.3389/fcell.2025.1592135

Figure Lengend Snippet: DKC1 expression is regulated by genomic alterations and female sex hormones but not by telomere length in UCEC tumors and cells. The TCGA cohort of UCEC tumors and UCEC-derived cells were analyzed (A) Differences in DKC1 expression in UCEC tumors carrying different copy numbers (B) Differences in DKC1 copy numbers between endometrial and serous/mixed types of UCEC tumors (C) The mutational landscape of the DKC1 gene in UCEC tumors (D) Up- and downregulation of DKC1 (Top panel) and MYC (Bottom panel) mRNA expression in UCEC-derived Ishikawa cells treated by 17 β-estradiol (left) and 1 nM MPA (right), respectively. *** and ****: P < 0.001 and 0.0001, respectively. Three independent experiments were performed (E) Correlation between DKC1 and estrogen receptor 1 (ESR1) (left) or PGR (right) expression (F) No correlation between DKC1 expression and the ratios of telomere length of UCEC tumors and corresponding patient blood cells. Telomere length of UCEC tumors and corresponding patient blood cells were obtained from reference Barthel FP, et al.

Article Snippet: Slides were blocked using 10% goat serum and incubated with a DKC1 polyclonal antibody (Cat 25420-1-AP, Proteintech, Rosemont, IL) for 2 h at room temperature.

Techniques: Expressing, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Comprehensive analyses of telomerase component DKC1 and its association with clinical, molecular and immune landscapes in uterine corpus endometrial carcinoma

doi: 10.3389/fcell.2025.1592135

Figure Lengend Snippet: Molecular features and pathway enrichments in DKC1-high UCEC tumors. A total of 545 tumors in the TCGA UCEC cohort were analyzed. Robustly increased Ki67 expression (A) , cell cycle score (B) , Stemness score (C) and EMT score (D) in DKC1-high tumors (E) The identification of enriched E2F and MYC targets as the hallmarks in DKC1-high tumors by GSEA analysis (F) The enriched cell cycle and DNA replication pathways in DKC1-high tumors by KEGG analysis.

Article Snippet: Slides were blocked using 10% goat serum and incubated with a DKC1 polyclonal antibody (Cat 25420-1-AP, Proteintech, Rosemont, IL) for 2 h at room temperature.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Comprehensive analyses of telomerase component DKC1 and its association with clinical, molecular and immune landscapes in uterine corpus endometrial carcinoma

doi: 10.3389/fcell.2025.1592135

Figure Lengend Snippet: DKC1 expression is associated with UCEC molecular subtypes and genomic aberrations. A total of 545 tumors in the TCGA UCEC cohort were analyzed (A) The association between DKC1 mRNA expression and molecular subtypes of UCECs (B–F) Comparisons of genomic alterations between DKC1-low and high tumors: Aneuploidy scores (B) , mitochondrial DNA (MTDNA) copies (C) , HRD (D) , MSI (E) and TMB (F) (G) Different frequencies of genomic alterations in important UCEC driver genes between DKC1-low and high tumors.

Article Snippet: Slides were blocked using 10% goat serum and incubated with a DKC1 polyclonal antibody (Cat 25420-1-AP, Proteintech, Rosemont, IL) for 2 h at room temperature.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Comprehensive analyses of telomerase component DKC1 and its association with clinical, molecular and immune landscapes in uterine corpus endometrial carcinoma

doi: 10.3389/fcell.2025.1592135

Figure Lengend Snippet: Identification of defective anti-tumor immunity and immunoexclusion microenvironments in DKC1-high tumors. A total of 544 tumors in the TCGA UCEC cohort were analyzed (A) Differences in immune, stromal and estimate scores between DKC1-high and low tumors, as determined using ESTIMATE analysis (B) TIDE analyses for comparison between DKC1-high and low tumors (C) CD274, CTLA4 and VTCN1 expression in DKC1-high and low tumors (D) Cancer immune cycle analyses of DKC1-high and low tumors. *, ** and ***: P < 0.05, 0.01 and 0.001, respectively (E) Differences in MHC scores between DKC1-high and low tumors (F) Prediction of DKC1-high and low tumors to immune checkpoint inhibitor sensitivity. Higher DKC1 expression is associated with lower sensitivity to immune checkpoint inhibitors.

Article Snippet: Slides were blocked using 10% goat serum and incubated with a DKC1 polyclonal antibody (Cat 25420-1-AP, Proteintech, Rosemont, IL) for 2 h at room temperature.

Techniques: Comparison, Expressing

Journal: BMC Biotechnology

Article Title: Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

doi: 10.1186/s12896-020-00653-8

Figure Lengend Snippet: Genes that were down-regulated (fold-change) in R-eCG-treated ovaries

Article Snippet: The PMSG ELISA kit was purchased from DRG International, Inc. (Mountain side, NJ, USA), Centriplus Centrifugal Filter Devices from Amicon Bio separations (Merck, Billerica, MA, USA), and an anti- myc antibody and antibodies against HSD17β1, ADAMTS1, Edn2, and OVGP1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques:

Journal: BMC Biotechnology

Article Title: Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

doi: 10.1186/s12896-020-00653-8

Figure Lengend Snippet: Localization of HSD17β1, ADAMTS1, EDN2, and OVGP1. The ovaries were induced to superovulate with 10 IU of either N-eCG or R-eCGβ/α, followed by 10 IU of hCG after 48 h. Representative immunohistochemical analyses for HSD17β1, ADAMTS1, EDN2, and OVGP1 were conducted with antisera, and a goat anti-rabbit IgG antibody (secondary antibody). According to the microarray and qRT-PCR results, HSD17β1 was up-regulated in the R-eCG-treated ovaries, while the other three proteins (ADAMTS1, EDN2, and OVGP1) were up-regulated in the N-eCG-treated ovaries. Immunohistochemistry was performed with a Vectastain ABC kit. Scale bar = 200 μm

Article Snippet: The PMSG ELISA kit was purchased from DRG International, Inc. (Mountain side, NJ, USA), Centriplus Centrifugal Filter Devices from Amicon Bio separations (Merck, Billerica, MA, USA), and an anti- myc antibody and antibodies against HSD17β1, ADAMTS1, Edn2, and OVGP1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunohistochemical staining, Microarray, Quantitative RT-PCR, Immunohistochemistry

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 1. Id1 Is a Target of the TbRI Inhibitor in PCTCs and in a Patient-Derived Mouse Model for Glioma (A) The genes were regulated by the treatment with 2 mM TbRI inhibitor for 3 hr in 11 human glioma PCTCs with a fold change over 1.4 or below 0.6 and a p < 0.001. (B) ID3, ID1, Smad7, and RhoB transcript levels were determined by qRT-PCR analysis. GAPDH RNA levels were used as an internal normalization control. *p < 0.05; **p < 0.001. Data are presented as means ± SD. (C and D) Cells from GBM1 and GBM2 neuro- spheres were inoculated in the brain of immuno- compromised mice. Twenty days after surgery, mice were orally treated twice a day with 100 mg/kg of TbRI inhibitor for 30 days. (C) Tumor area was quantified. (D) Images from the entire mouse brains were obtained by MRI (arrowheads indicate tumors). (E) Immunohistochemistry for Id1 (upper panel) and double immunofluorescence for Id1 and CD31 (middle panel) were performed in tumors generated by GBM neurospheres. For immunoflu- orescence, nuclei were counterstained with Hoechst. Scale bar, 100 mm. Representative images are shown and percentage of Id1-positive cells was calculated (lower panel). Data are pre- sented as means ± SD. (F) Cells from GBM1 neurospheres were inocu- lated in the brain of immunocompromised mice. Forty days after surgery, mice were orally treated twice a day with 100 mg/kg of TbRI inhibitor for 10 days. Subsequently, 10 mm slides from mouse brains were stained with H&E and tumors were localized and microdissected. ID1 tran- script levels were determined by qRT-PCR (n = 4 for vehicle; n = 3 for TbRI inhibitor). Data are presented as means ± SD. See also Fig- ure S1.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Derivative Assay, Quantitative RT-PCR, Control, Immunohistochemistry, Generated, Staining

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 4. Patient-Derived Neurospheres Have a CD44high/Id1high Cell Population (A) Cells from GBM1 neurospheres were dissociated and seeded on coverslips. Immunocytochemistry for Id1and CD44 was performed and nuclei were counter- stained with Hoechst. Scale bar, 10 mm. Quantification of Id1 and CD44 expression levels per cell is shown. (B) FACS analysis of CD44 levels was performed in GBM neurospheres (upper panel). Isotype control is shown. Cells with high or low levels of CD44 from the indicated GBM neurospheres were sorted by FACS and CD44, ID1, ID2, and ID3 transcript levels were determined by qRT-PCR (lower panels). *p < 0.01; **p < 0.001 compared to CD44low. Data are presented as means ± SD. (C) Cells from GBM1 neurospheres were sorted by FACS according to CD44 levels, and the levels of Id1, Id3, and tubulin were determined by immunoblotting. See also Figure S4.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Derivative Assay, Immunocytochemistry, Staining, Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 6. Id Proteins Mediate the Effect of TGF-b on the CD44high Population and the Oncogenic Potential of GBM Neurospheres (A) Cells from GBM1 were infected with the indicated lentivirus. Neurospheres were treated with 100 pM TGF-b1 for 3 hr, 2 mM TbRI inhibitor for 8 hr, or left untreated, and the levels of Id1, Id3, p-Smad2, Smad2, and tubulin were determined by immunoblotting. (B) Cells from GBM1 neurospheres previously infected with the indicated lentivirus were treated with 100 pM TGF-b or 2 mM TbRI inhibitor for 7 days or left untreated. FACS analysis of CD44 levels (left panel) and quantification of the frequency of CD44high cells are shown (right panel). Bars represent the percent of CD44high cells in untreated, TGF-b-treated, and TbRI inhibitor-treated lentiviral-infected neurospheres, as indicated. Data are presented as means. (C–E) Cells from GBM1 neurospheres previously infected with the indicated lentivirus were treated for 7 days with 2 mM TbRI inhibitor, or left untreated. Subse- quently, cells were dissociated, counted, and equal numbers of cells were inoculated in the brain of NOD-SCID mice. (C) Images from the entire mouse brains were obtained by MRI. Arrowheads indicate tumors. (D) Tumor area was quantified (p = 0.004 comparing mice inoculated with untreated neurospheres with mice inoculated with neurospheres treated with the TbRI inhibitor; p = 0.002 comparing mice inoculated with neurospheres infected with the lentivirus control with mice inoculated with neurospheres infected with the sh2 ID1/ID3). (E) Tumor incidence was determined. Data are presented as means ± SD. (F–H) Cells from GBM1 neurospheres previously infected with the indicated lentivirus were treated during 7 days with 2 mM TbRI inhibitor or left untreated. Subse- quently, cells were dissociated, counted, and equal numbers of cells were inoculated in the brain of immunocompromised mice. (F) Images from the entire mouse brains were obtained by MRI. Arrowheads indicate tumors. (G) Tumor area was quantified (p = 0.04 comparing mice inoculated with control neurospheres with mice inoculated with Id1 overexpressing neurospheres), and (H) tumor incidence was determined. Data are presented as means ± SD. See also Figure S6.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Infection, Western Blot, Control

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 7. TbRI Inhibitor Decreases Id1, Id3, and CD44 Levels in Tumors and Prevents Tumor Recurrence in Vivo (A) Scheme showing the experimental procedure. (B and C) Cells from GBM1 neurospheres were inoculated in the brain of immunocompromised mice. Forty days after surgery, mice were orally treated twice a day with 100 mg/kg of TbRI inhibitor for 10 days. Subsequently, human tumor cells were isolated from the brain of mice treated or untreated with TbRI inhibitor through sorting of human MHC-I-positive cells. (B) ID1, ID3, and CD44 transcripts levels were determined by qRT-PCR, and (C) CD44 levels were assessed by FACS. Isotype control is shown. Data are presented as means ± SD. (D and E) Human tumor cells obtained from mice treated or untreated with the TbRI inhibitor were inoculated in the brain of secondary mice. (D) Thirty days after surgery, images from the entire mouse brains were obtained by MRI, tumor area was quantified (n = 4 mice inoculated with cells from untreated mice; n = 8 mice inoculated with cells from TbRI inhibitor treated mice), and (E) tumor incidence was determined. Data are presented as means ± SD.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: In Vivo, Isolation, Quantitative RT-PCR, Control

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 8. Id1 and CD44 Coexpression Correlates with Overall Survival in GBM Patients (A) A tissue microarray of 43 different GBM patients was stained with an antibody against Id1 and the frequency of Id1-positive nuclei was calculated. (B) CD44/Id1 and CD31/Id3 double immunofluorescence of samples from GBM patients. Magnification of the indicated areas stained with Id1/CD44 and Id3/ CD31 are shown in the right panels. Nuclei were counterstained with Hoechst. Scale bar, 50 mm. (C) CD44, Id1, and CD31 immunohistochemistry of samples from GBM patients. Scale bar, 100 mm. (D) Kaplan-Meier curves showing that the overall survival of patients with both ID1 mRNA levels upregulated R3-fold and CD44 mRNA levels upregulated R10-fold is significantly lower than the rest of the patients (p = 0.03) by log-rank test. Data obtained from the Repository for Molecular Brain Neoplasia Data (REMBRANDT) program form the National Cancer Institute. See also Figure S7.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Microarray, Staining, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas

doi: 10.1186/1756-9966-31-85

Figure Lengend Snippet: Genes identified by SEREX

Article Snippet: Immunohistochemistry with the polyclonal antibody against SH3GL1 (sc-25495; Santa Cruz) was performed using commercially available reagents, Histofine (Nichirei Bioscience Inc, Tokyo, Japan), and according to the manufacturer’s recommendations.

Techniques: Sequencing, Recombinant, Amplification, Binding Assay, Coagulation, Variant Assay, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas

doi: 10.1186/1756-9966-31-85

Figure Lengend Snippet: The increasing levels of antibodies to SH3GL1 in sera of the patients with low-grade glioma. Serum antibody level to SH3GL1 was examined by the ELISA as described in the legends of Figure . First screening test (A) and the individual validation test (B) , revealed the significant higher levels of autologous antibody against SH3GL1 in low-grade glioma patients, than healthy donors (P = 0.045 and 0.0189).

Article Snippet: Immunohistochemistry with the polyclonal antibody against SH3GL1 (sc-25495; Santa Cruz) was performed using commercially available reagents, Histofine (Nichirei Bioscience Inc, Tokyo, Japan), and according to the manufacturer’s recommendations.

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas

doi: 10.1186/1756-9966-31-85

Figure Lengend Snippet: Kaplan-Meier analysis for the overall survival of the patients with low-grade gliomas according to the serum level of anti-SH3GL1 autoantibody. The patients with higher serum level of anti-SH3GL1 autoantibody (solid line) survived significantly longer than those with lower levels (gray line) (p = 0.0124).

Article Snippet: Immunohistochemistry with the polyclonal antibody against SH3GL1 (sc-25495; Santa Cruz) was performed using commercially available reagents, Histofine (Nichirei Bioscience Inc, Tokyo, Japan), and according to the manufacturer’s recommendations.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas

doi: 10.1186/1756-9966-31-85

Figure Lengend Snippet: Comparison of serum antibody levels among deletion mutants of SH3GL1. To confirm the epitope site, some SH3GL1 deletion mutants (A) were synthesized. Serum antibody levels were examined by ELISA with SH3GL1 muta-1 (B) , mut-2 (C) , mut-3 (D) and mut-4 (E) , and the 10–20 amino acids at the C-terminal end were indicated as the epitope site.

Article Snippet: Immunohistochemistry with the polyclonal antibody against SH3GL1 (sc-25495; Santa Cruz) was performed using commercially available reagents, Histofine (Nichirei Bioscience Inc, Tokyo, Japan), and according to the manufacturer’s recommendations.

Techniques: Comparison, Synthesized, Enzyme-linked Immunosorbent Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas

doi: 10.1186/1756-9966-31-85

Figure Lengend Snippet: The detection of epitope site by overlapped peptide array. Series of peptides of 14 amino acid residues, composed of SH3GL1, were synthesized with overlapping by 12 amino acids, and were blotted in nitrocellulose membranes using F-moc amino acids (A) . Three sera of the patients with low-grade glioma indicated the fine reaction in spot 177 and 178 (C) , compared to two normal volunteers (D) and no serum control (B) . The calculated fluorescence intensity, normalized by background control, revealed that these spots were suggested as a minimum epitope site (E).

Article Snippet: Immunohistochemistry with the polyclonal antibody against SH3GL1 (sc-25495; Santa Cruz) was performed using commercially available reagents, Histofine (Nichirei Bioscience Inc, Tokyo, Japan), and according to the manufacturer’s recommendations.

Techniques: Peptide Microarray, Synthesized, Control, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas

doi: 10.1186/1756-9966-31-85

Figure Lengend Snippet: Immunohistochemical analysis of SH3GL1 in glioma cells. Immunohistochemical stain for SH3GL1 in whole normal brain, consisted of white matter and gray matter (A) , and three representative results of normal white matter, low-grade glioma and high-grade glioma (B) were shown. Immunostaining for SH3GL1 was classified in five groups, and numbers of tissues in each group were scored (C).

Article Snippet: Immunohistochemistry with the polyclonal antibody against SH3GL1 (sc-25495; Santa Cruz) was performed using commercially available reagents, Histofine (Nichirei Bioscience Inc, Tokyo, Japan), and according to the manufacturer’s recommendations.

Techniques: Immunohistochemical staining, Staining, Immunostaining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas

doi: 10.1186/1756-9966-31-85

Figure Lengend Snippet: Changes in the serum autoantibody level to SH3GL1 in a rat brain tumor model using C6 rat glioblastoma cells which were confirmed to express SH3GL1 protein. MRI studies show a steady growth of tumor mass in the rat brain (A) . The serum autoantibody levels were significantly increased at 2-week after tumor inoculation, and tended to decrease at 4-week after the inoculation (B).

Article Snippet: Immunohistochemistry with the polyclonal antibody against SH3GL1 (sc-25495; Santa Cruz) was performed using commercially available reagents, Histofine (Nichirei Bioscience Inc, Tokyo, Japan), and according to the manufacturer’s recommendations.

Techniques:

Journal: Journal of Thoracic Disease

Article Title: SH3BGRL2 as a vital tumor suppressor and prognostic factor in human esophageal squamous cell carcinoma

doi: 10.21037/jtd-2025-1878

Figure Lengend Snippet: mRNA and protein expression levels and prognostic significance of SH3BGRL2 in ESCC. (A) Differentially expressed mRNAs between ESCC tumor samples and adjacent normal tissues identified by RNA sequencing (T: tumor tissue; N: normal tissue). (B,C) SH3BGRL2 expression levels in ESCC as analyzed via the GEPIA database (P<0.01) and GEO database (both P<0.05, GSE23400, GSE17351, and GSE45670). (D) Representative tissue microarray images of SH3BGRL2 staining via immunohistochemistry (×200): positive SH3BGRL2 expression in tumor tissues (a) and normal tissues (b) and negative SH3BGRL2 expression in tumor tissues (c) and normal tissues (d). (E) Percentages of SH3BGRL2-positive samples in tumor and nontumor esophageal tissues (31.2% vs. 51.0%; P<0.001). (F,G) Kaplan-Meier curves showing the disease-free survival (F) or overall survival (G) of patients with ESCC and higher SH3BGRL2 expression and in those with lower SH3BGRL2 expression. Error bars represent the standard deviation of the mean. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 (Student t -test, one-way ANOVA). ANOVA, analysis of analysis; DFS, disease-free survival; ESCC, esophageal squamous cell carcinoma; GEO, Gene Expression Omnibus; GEPIA, Gene Expression Profiling Interactive Analysis; OS, overall survival; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2.

Article Snippet: Standard IHC was performed with a primary antibody against human SH3BGRL2 (#21944-1-AP; Proteintech, Rosemont, IL, USA) at a dilution of 1:200.

Techniques: Expressing, RNA Sequencing, Microarray, Staining, Immunohistochemistry, Standard Deviation, Gene Expression, Binding Assay

Journal: Journal of Thoracic Disease

Article Title: SH3BGRL2 as a vital tumor suppressor and prognostic factor in human esophageal squamous cell carcinoma

doi: 10.21037/jtd-2025-1878

Figure Lengend Snippet: SH3BGRL2 inhibited the proliferation of ESCC PDCs. (A) RNA sequencing and western blot analysis of SH3BGRL2 expression levels in different ESCC PDCs. Western blot assays validated the efficiencies of SH3BGRL2 knockdown in ZEC043, ZEC056, and ZEC145 cells (B,C) and overexpression in ZEC014 cells (D). CCK-8 and colony formation with crystal violet staining assays analyzed cell proliferation in ZEC043, ZEC056, ZEC145 cells (E-H), and ZEC014 cells (I). Each picture represents a well of a 6-well plate. Data are presented as the mean ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001 (Student t -test). The grayscale analysis values of western blot bands are listed below the bands. CCK-8, Cell Counting Kit-8; ESCC, esophageal squamous cell carcinoma; OD, optical density; PDC, patient-derived cell line; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2.

Article Snippet: Standard IHC was performed with a primary antibody against human SH3BGRL2 (#21944-1-AP; Proteintech, Rosemont, IL, USA) at a dilution of 1:200.

Techniques: RNA Sequencing, Western Blot, Expressing, Knockdown, Over Expression, CCK-8 Assay, Staining, Standard Deviation, Cell Counting, Derivative Assay, Binding Assay

Journal: Journal of Thoracic Disease

Article Title: SH3BGRL2 as a vital tumor suppressor and prognostic factor in human esophageal squamous cell carcinoma

doi: 10.21037/jtd-2025-1878

Figure Lengend Snippet: SH3BGRL2 suppressed the growth of ESCC PDCs in vivo . (A) Representative images of BALB/c nude mice subcutaneously injected with vector control (upper row) or SH3BGRL2 knockdown ZEC-145 cells (lower row). (B) Analysis of tumor volume of mice measured weekly (n=8 per group). (C) Analysis of tumor weight of xenograft tumors 4 weeks after tumor inoculation (n=8 per group). Data are presented as the mean ± standard deviation. *, P<0.05 (Student t -test and Chi-squared test). ESCC, esophageal squamous cell carcinoma; PDC, patient-derived cell line; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2.

Article Snippet: Standard IHC was performed with a primary antibody against human SH3BGRL2 (#21944-1-AP; Proteintech, Rosemont, IL, USA) at a dilution of 1:200.

Techniques: In Vivo, Injection, Plasmid Preparation, Control, Knockdown, Standard Deviation, Derivative Assay, Binding Assay

Journal: Journal of Thoracic Disease

Article Title: SH3BGRL2 as a vital tumor suppressor and prognostic factor in human esophageal squamous cell carcinoma

doi: 10.21037/jtd-2025-1878

Figure Lengend Snippet: SH3BGRL2 inhibited the EGR1 expression of ESCC cells. (A) Differentially expressed genes between SH3BGRL2-silenced and vector control ZEC145 cells. (B) Elevated mRNA expression of EGR1 in SH3BGRL2-silenced ZEC145 and vector control cells, as indicated in orange borders. (C) Transcription factors associated with differentially expressed genes between SH3BGRL2-silenced and vector control ZEC145 cells. Results show the significant enrichment of the C2H2 zinc finger transcription factor family (zf-C2H2). X-axis: the number of genes in each transcription factor family. (D) The significant negative correlation between EGR1 and SH3BGRL2 expression in ESCC tissues as analyzed via TCGA database (P<0.001). (E) Quantitative reverse transcription-PCR confirmed that EGR1 mRNA expression was increased in SH3BGRL2-knockdown cells (P<0.001). (F,G) Protein expression of EGR1 in SH3BGRL2-silenced ZEC145 cells and SH3BGRL2-overexpressing ZEC014 cells and corresponding vector control cells. Data are presented as the mean ± standard deviation. The grayscale analysis values of western blot bands are listed below the bands. **, P<0.01; ***, P<0.001 (Student t -test). EGR1, early growth response 1; ESCC, esophageal squamous cell carcinoma; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2; TCGA, The Cancer Genome Atlas; TPM, transcripts per million.

Article Snippet: Standard IHC was performed with a primary antibody against human SH3BGRL2 (#21944-1-AP; Proteintech, Rosemont, IL, USA) at a dilution of 1:200.

Techniques: Expressing, Plasmid Preparation, Control, Reverse Transcription, Knockdown, Standard Deviation, Western Blot, Binding Assay