21972 1 ap  (Proteintech)

Journal: Journal of Pain Research

Article Title: Metabolomics Analysis Reveals Gut Microbiota-Associated Sakuranin Modulates Endometrial Stem Cell Differentiation and Inflammation to Alleviate Pain in Endometriosis

doi: 10.2147/JPR.S557430

Figure Lengend Snippet: Sakuranin inhibits the differentiation of EnSCs into mesenchymal cells and reduces the levels of inflammatory factors. ( A ) The expression level of the Sushi Domain-containing 2 (SUSD2) protein in EnSCs. The expression of the SUSD2 protein is presented in red fluorescence, and the nuclear DAPI staining is shown in blue; ( B ) The flow cytometry scatter plot of EnSCs without treatment with the SUSD2 antibody (left) and of EnSCs incubated with the SUSD2 antibody (right); ( C ) Screening for the optimal concentration of sakuranin; ( D – F ) Detection of the contents of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in EnSCs by enzyme-linked immunosorbent assay (ELISA); ( G – I ) Detection of the expression levels of the activated leukocyte cell adhesion molecule (ALCAM) and the vascular endothelial growth factor (VEGF) in EnSCs by Western blot and the quantitative analysis of protein expression; ( J – M ) Detection of the mRNA expressions of epithelial cadherin (E-cadherin), cytokeratin 10 (Cytokeratin 10), vimentin, and neural cadherin (N-cadherin) in human primary EnSCs by real-time quantitative polymerase chain reaction (RT-qPCR); ( N ) Phalloidin staining of the cytoskeleton of EnSCs. The green fluorescence represents phalloidin staining, and the blue fluorescence represents the counterstaining of the nucleus by DAPI; ( O ) Detection of the migratory ability by scratch assay and the quantitative analysis of the scratch healing of EnSCs. The cells were treated with 1 μg/mL lipopolysaccharide (LPS) for 48 hours. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After the transfer, the membranes were blocked at room temperature for 2 hours before overnight incubation with primary antibodies, including VEGF (1:5000, 19003-1-AP, Proteintech); ALCAM (1:5000, 21972-1-AP, Proteintech); GAPDH (1:15000, 60004-1-lg, Proteintech) at 4 °C.

Techniques: Expressing, Fluorescence, Staining, Flow Cytometry, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Wound Healing Assay

Journal: Journal of Pain Research

Article Title: Metabolomics Analysis Reveals Gut Microbiota-Associated Sakuranin Modulates Endometrial Stem Cell Differentiation and Inflammation to Alleviate Pain in Endometriosis

doi: 10.2147/JPR.S557430

Figure Lengend Snippet: Immunofluorescence detection of protein expression in the endometrium of normal mice and the ectopic endometrial tissue of EMS mice. ( A – D ) Tissue immunofluorescence detection of the expressions of Cluster of differentiation 146 (CD146), ALCAM, VEGF, and Ki67 proteins. The expressions of the detected proteins are presented in red fluorescence, and the nuclear DAPI staining is shown in blue; ( E ) Tissue immunofluorescence detection of the expressions of tyrosine hydroxylase (TH) and the marker substance P (SP). Red represents the expression of SP protein, green represents the expression of TH protein, and blue represents the nuclear staining by DAPI.

Article Snippet: After the transfer, the membranes were blocked at room temperature for 2 hours before overnight incubation with primary antibodies, including VEGF (1:5000, 19003-1-AP, Proteintech); ALCAM (1:5000, 21972-1-AP, Proteintech); GAPDH (1:15000, 60004-1-lg, Proteintech) at 4 °C.

Techniques: Immunofluorescence, Expressing, Fluorescence, Staining, Marker

Journal: eLife

Article Title: Single-cell profiling reveals the intratumor heterogeneity and immunosuppressive microenvironment in cervical adenocarcinoma

doi: 10.7554/eLife.97335

Figure Lengend Snippet: ( A ) Circle plots showing the interacting networks between epithelial cell sub-clusters and T cell sub-clusters via the pathway of ALCAM by comparing adenocarcinoma (ADC) and squamous cell carcinoma (SCC). ( B ) Circle plots simplified from ( A ) to show the interactions among target cell clusters via ALCAM pathway. ( C ) Circle plots showing the interacting networks between epithelial cell sub-clusters and T cell sub-clusters via the pathway of MHC-II by comparing ADC and SCC. ( D ) Circle plots simplified from ( C ) to show the interactions among target cell clusters via MHC-II pathway. From ( A ) to ( D ), the direction of each arrow shows the regulation from outputting cells to incoming cells. The width of the each line shows the predicted weight and strength of regulation. ( E ) Bubble plot showing the probability of ligand-to-receptor combination of each pathway between two different target sub-clusters of cells by comparing ADC with SCC. ( F ) Circle plots showing Tregs regulate epithelial cells via the TGF-β pathway, which is solely activated in ADC. ( G ) Bubble plot showing the probability of ligand-to-receptor combination of TGF-β pathway between Tregs and epithelial cells by comparing ADC with SCC. The pathways of ADGRE5, CD46, GZMA, and NAMPT are used as negative controls. ( H ) Dual immunofluorescence (IF) staining confirming that in SLC26A3 high regions of CC tissues, more FOXP3 + cells are recruited than in SLC26A3 low regions (left). The numbers of recruited FOXP3 + cell are quantified using histogram plot (right). Three individual samples with ROI were calculated and p<0.01 were marked with **, showing significant difference. ( I ) Multiplexed IF staining confirming the interaction between CD6 (on FOXP3 + cells) and ALCAM (on SLC26A3 high epithelial cells) in the ALCAM pathway. ( J ) Multiplexed IF staining showing that the recruitment of FOXP3 + cells toward SLC26A3 high cells might induce EMT (marked with E-cadherin) and increase the stemness (marked with ALDH1A1) of tumor cells, via TGF-β pathway.

Article Snippet: Antibody , ALCAM polyclonal antibody , Proteintech , Cat# 21972-1-AP , .

Techniques: Immunofluorescence, Staining

Journal: eLife

Article Title: Single-cell profiling reveals the intratumor heterogeneity and immunosuppressive microenvironment in cervical adenocarcinoma

doi: 10.7554/eLife.97335

Figure Lengend Snippet:

Article Snippet: Antibody , ALCAM polyclonal antibody , Proteintech , Cat# 21972-1-AP , .

Techniques: Recombinant

Journal: Frontiers in Oncology

Article Title: USP7 regulates growth and maintains the stemness of p53-mutant colorectal cancer cells via stabilizing of mutant p53

doi: 10.3389/fonc.2024.1427663

Figure Lengend Snippet: USP7 is highly expressed in colorectal cancer stem-like cells. (A) Protein expression levels of USP7, p53, CD133, CD166 and CD44 were tested in spheroid cells and adherent cells by western blotting. (B) Quantification of indicated protein levels in (A) by NIH ImageJ software. (C) mRNA expression levels of USP7 and TP53 were determined by qPCR. All values represented the mean ± SD (n = 3).The significance was determined by student’s t test (**P< 0.01 and ***P< 0.001 vs. control).

Article Snippet: The transferred PVDF membranes were incubated with the following primary antibody at room temperature for 3 h: USP7 and p53 (primary antibody: 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), CD133, CD166, CD44 and GAPDH (primary antibody: 1:1000, Proteintech, Rosemont, IL).

Techniques: Expressing, Western Blot, Software, Control

Journal: Frontiers in Oncology

Article Title: USP7 regulates growth and maintains the stemness of p53-mutant colorectal cancer cells via stabilizing of mutant p53

doi: 10.3389/fonc.2024.1427663

Figure Lengend Snippet: P5091 reduces the population of CSCs and inhibits cell migration capacity of colorectal cancers. (A) MTS assay were used to determine the growth inhibition effect of P5091 on cancer cells. (B) Cells were cultured under ultralow-attachment conditions for 7 days to enrich CSCs, and then treated with P5091 for 24 h (original magnification, 10× or 20×). (C) The spheroids size of 1st spheroid formation (upper) and 2nd spheroid formation (down) of HCT116 and SW480 cells were determined (original magnification, 20×). (D) The protein levels of CD133, CD166, CD44 and OCT4 were determined via western blot. (E) Quantification of indicated protein levels in (D) were detected by NIH ImageJ software. The values represented the mean ± SD (n = 3). (F) The mRNA levels of SOX2 , OCT4 and NANOG were determined by qPCR, GAPDH mRNA was used as an internal control. (G, H) Wound closure of HCT116 and SW480 cells in the presence or absence of P5091. Data are shown as means ± SD. The significance was determined by student’s t test (*P < 0.05, **P < 0.01 and ***P < 0.001 vs. control).

Article Snippet: The transferred PVDF membranes were incubated with the following primary antibody at room temperature for 3 h: USP7 and p53 (primary antibody: 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), CD133, CD166, CD44 and GAPDH (primary antibody: 1:1000, Proteintech, Rosemont, IL).

Techniques: Migration, MTS Assay, Inhibition, Cell Culture, Western Blot, Software, Control

Journal: Frontiers in Oncology

Article Title: USP7 regulates growth and maintains the stemness of p53-mutant colorectal cancer cells via stabilizing of mutant p53

doi: 10.3389/fonc.2024.1427663

Figure Lengend Snippet: Downregulation of USP7 weakens cell self-renewal of CSCs. (A) Growth curves depicting the slow proliferation of HCT116, SW480, SKBR3 and MDA-MB-468 cells with USP7 interference. Cell proliferation was assessed by MTS assay and colony formation assay. USP7 protein levels were confirmed by western blot. (B) Comparison of sphere formation the absence or presence of USP7. (original magnification, 10× or 20×). USP7 protein levels were confirmed by western blot. The USP7 and GAPDH blots of MDA-MB-468 between (A, B) were reused. (C) The protein levels of CD133, CD166 and CD44 were determined via western blot. (D) The mRNA levels of USP7, SOX2, OCT4 and NANOG were determined by qPCR. GAPDH mRNA was used as an internal control. (E) Wound closure among normal and USP7 knockdown cell lines at 48 h (F) Comparison of sphere formation among normal and USP7 overexpression cell lines (original magnification, 10×, 20× and 40×). USP7 levels were tested by western blot. Quantification of all indicated protein levels by NIH ImageJ software. The values represented the mean ± SD (n = 3). The significance was determined by student’s t test (**P < 0.01 and ***P < 0.001 vs. control).

Article Snippet: The transferred PVDF membranes were incubated with the following primary antibody at room temperature for 3 h: USP7 and p53 (primary antibody: 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), CD133, CD166, CD44 and GAPDH (primary antibody: 1:1000, Proteintech, Rosemont, IL).

Techniques: MTS Assay, Colony Assay, Western Blot, Comparison, Control, Knockdown, Over Expression, Software