Journal: Molecular & Cellular Proteomics : MCP

Article Title: Seminal Plasma Proteome as an Indicator of Sperm Dysfunction and Low Sperm Motility in Chickens

doi: 10.1074/mcp.RA120.002017

Figure Lengend Snippet: Western blot analysis of seminal IL6ST, CKMT2, GAPDH and CYC abundance in low and high motility semen samples. A, Western blotting showing protein abundances of IL6ST, CKMT2, GAPDH and CYC in seminal plasma of LSM and HSM; M, pre-stained protein ladder; L1-L4, samples of low sperm motility group; H1-H4, samples of high sperm motility group. B, Bars represent relative protein quantification of IL6ST; C, Bars represent relative protein quantification of GAPDH; Protein quantification was normalized to the mean of HSM; Values are specified as mean ± S.E.; LSM, low sperm motility; HSM, high sperm motility; * p < 0.05, ** p < 0.01.

Article Snippet: The membranes were incubated with the primary antibodies of 1: 5000 diluted anti-CKMT2 (Rabbit Polyclonal, 13207–1-AP, Proteintech, Wuhan, Hubei, China), 1: 2000 diluted anti-CYC (Rabbit Polyclonal, bs-0013R, Bioss, Beijing, China), 1:10000 diluted anti-GAPDH (Rabbit Polyclonal, 10494–1-AP, Proteintech), or 1:2000 diluted anti-IL6ST (Rabbit Polyclonal, bs-5151R, Bioss).

Techniques: Western Blot, Staining

Journal: Molecular Cancer Research

Article Title: OLR1 Promotes Pancreatic Cancer Metastasis via Increased c-Myc Expression and Transcription of HMGA2

doi: 10.1158/1541-7786.mcr-19-0718

Figure Lengend Snippet: Figure 4. OLR1 increases the metastasis of pancreatic cancer cells by regulating c-Myc and HMGA2. A, Analysis of gene expression profiles identified 26 genes positively correlated with OLR1 levels and 39 genes negatively correlated with OLR1 levels in Bxpc-3 cells. B, Cluster heatmap of mRNA expression profiles in Bxpc-3 cells with OLR1 overexpression or knockdown. C and D, RT-qPCR of c-Myc (C) and HMGA2 mRNA (D) in Bxpc-3 cells. GAPDH mRNA served as a loading control. E, Western blot analysis of c-Myc and HMGA2 in Bxpc-3 cells. F and G, c-Myc (F) and HMGA2 (G) expression positively correlated with OLR1 expression in the TCGA database of patients with pancreatic cancer (n ¼ 178). H–K, Rescue assayswere performed in invitro transwell and invasion migration experiments in Bxpc-3 cells. Representative images are shown. Magnification, 100. Cell numbers of migrated and invasive cells are shown in the right panels (, P < 0.05; , P < 0.01; , P < 0.001).

Article Snippet: Primary antibodies targeting OLR1 and HMGA2 were purchased from Proteintech Group and c-Myc and b-actin antibodies were obtained from Cell Signaling Technology. qRT-PCR TRizol reagent (Invitrogen) was used for the extraction of total RNA and cDNA synthesis was performed using the Takara First Strand cDNA Synthesis Kit following the manufacturer's instructions. qRT-PCR assays were performed independently at least three times.

Techniques: Gene Expression, Expressing, Over Expression, Knockdown, Quantitative RT-PCR, Control, Western Blot, Migration

Journal: Molecular Cancer Research

Article Title: OLR1 Promotes Pancreatic Cancer Metastasis via Increased c-Myc Expression and Transcription of HMGA2

doi: 10.1158/1541-7786.mcr-19-0718

Figure Lengend Snippet: Figure 5. c-Myc promotes the transcription of HMGA2. A and B, The expression of HMGA2 regulated by c-Myc was validated in Bxpc-3 cells by RT-qPCR (A) and Western blot analysis (B). C, HMGA2 expression positively correlated with c-Myc expression in the TCGA database of patients with pancreatic cancer (r ¼ 0.24; P < 0.001). D, Schematic of the HMGA2 gene promoter. Two potential c-Myc binding elements and their respective locations (top) and the design of mutants for each binding element are shown (middle and bottom). E, Evaluation of ENCODE c-Myc ChIP-sequencing data. Peaks represent c-Myc enrichment at the HMGA2 promoter in immortalized MCF10A mammary epithelial cells, HepG2 hepatocellular carcinoma, and Gm12878 lymphocyte cells. F and G, ChIP with antibodies against c-Myc or isotype control IgG and qRT-PCR for c-Myc binding element 1 (F) and 2 (G) was performed in 293T, Bxpc-3, and Mia PaCa-2 cells. H, Dual luciferase assay was performed in 293T, Bxpc-3, and Mia PaCa-2 cells with c-Myc overexpression and control cells transfected with pGL3 empty vector, pGL3-HMGA2 WT, pGL3-HMGA2- Mut1, or pGL3-HMGA2-Mut2. The results are shown as relative luciferase activity to pGL3 empty vector in corresponding cells after normalization with Renilla luciferase activity (, P < 0.05; , P < 0.01; , P < 0.001).

Article Snippet: Primary antibodies targeting OLR1 and HMGA2 were purchased from Proteintech Group and c-Myc and b-actin antibodies were obtained from Cell Signaling Technology. qRT-PCR TRizol reagent (Invitrogen) was used for the extraction of total RNA and cDNA synthesis was performed using the Takara First Strand cDNA Synthesis Kit following the manufacturer's instructions. qRT-PCR assays were performed independently at least three times.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Binding Assay, ChIP-sequencing, Control, Luciferase, Over Expression, Transfection, Plasmid Preparation, Activity Assay

Journal: Molecular Cancer Research

Article Title: OLR1 Promotes Pancreatic Cancer Metastasis via Increased c-Myc Expression and Transcription of HMGA2

doi: 10.1158/1541-7786.mcr-19-0718

Figure Lengend Snippet: Figure 6. OLR1 upregulates HMGA2 through c-Myc to promote pancreatic cancer cell metastasis, and combined OLR1, HMGA2, and c-Myc are associated with poor prognosis of patients with pancreatic cancer. A and B, Rescue assays were performed with in vitro transwell and invasion experiments in Bxpc-3 cells. Representative images are shown. Magnification, 100; numbers of migrated and invasive cells are shown in the right panels. C, E-cadherin, Snail, and b-catenin level were assessed by Western blot analysis in Bxpc-3 and Mia PaCa-2 cells with HMGA2 overexpression or knockdown. D, c-Myc and HMGA2 protein levels were assessed in Bxpc-3 cells by rescue assay using Western blot analysis. E, Representative images of IHC staining of OLR1, c-Myc, and HMGA2 in the same pancreatic cancer tissue. All proteins are expressed at low levels in the two cases on the left and overexpressed in the two cases on the right. Magnification, 50; scale bars, 200 mm. The images in the top right panels represent magnified views of the boxed regions. Magnification, 200; scale bars, 100 mm. F–H, IHC score analysis results revealed that OLR1, c-Myc, and HMGA2 positively correlated with each other. I and J, Kaplan–Meier prognosis analysis of patients with pancreatic cancer with combined OLR1, c-Myc, and HMGA2 in TMA (H) and TCGA database (I). Group 1, one protein (OLR1, c-Myc, or HMGA2) is highly expressed; Group 2, two proteins (OLR1, c-Myc, and/or HMGA2) are highly expressed; Group 3, all three proteins (OLR1, c-Myc, and HMGA2) are highly expressed (, P < 0.05; , P < 0.01; , P < 0.001).

Article Snippet: Primary antibodies targeting OLR1 and HMGA2 were purchased from Proteintech Group and c-Myc and b-actin antibodies were obtained from Cell Signaling Technology. qRT-PCR TRizol reagent (Invitrogen) was used for the extraction of total RNA and cDNA synthesis was performed using the Takara First Strand cDNA Synthesis Kit following the manufacturer's instructions. qRT-PCR assays were performed independently at least three times.

Techniques: In Vitro, Western Blot, Over Expression, Knockdown, Rescue Assay, Immunohistochemistry

Journal: iScience

Article Title: Ovulation provides excessive coagulation and hepatocyte growth factor signals to cause postoperative intraabdominal adhesions

doi: 10.1016/j.isci.2024.109788

Figure Lengend Snippet:

Article Snippet: The membranes were then blocked with 5% non-fat milk powder in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) for one hour followed by incubation with primary antibodies such as anti-MET (BS-0668R, Bioss), anti-p-MET (#3077, Cell Signaling), and anti-actin (#4970, Cell Signaling).

Techniques: Recombinant, Staining, Software

Journal: bioRxiv

Article Title: Mechanical Ventilation induced DNA Damage and P21 in an Acute Aging Model of Lung Injury

doi: 10.1101/2022.03.08.483505

Figure Lengend Snippet: Immunofluorescence staining of lung section for both young and old mice. Staining for KRT8 (red), a marker of transiently differentiated AT2, and SFTPC (green), an AT2 marker. DAPI (blue) was used as a counterstain. The staining reveals an increase in KRT8 positive cells with age and mechanical ventilation and a decrease in SFTPC in all groups compared to young non-ventilated. NV: non-ventilated and HP: high pressure.

Article Snippet: Primary antibodies: mouse anti-gamma H2AX (Novus Biologicals, NB100-74435), rabbit anti-Ki67/MKi67 (Novus Biologicals, NB500-170SS), rabbit anti-SP-C (Bioss, bs-10067R), rat anti-KRT8 (CreativeBiolabs, CBDH1469).

Techniques: Immunofluorescence, Staining, Marker