rabbit polyclonal anti cd133  (Proteintech)

Journal: Frontiers in Immunology

Article Title: TREM1 is essential for maintaining stemness of liver cancer stem-like cells in hepatocellular carcinoma

doi: 10.3389/fimmu.2025.1618342

Figure Lengend Snippet: TREM1 promotes tumorigenicity, clonogenic potential and spheroid formation in CD133 + EpCAM + liver cancer stem-like cells. (A) Flow cytometry dot plots reveal significant TREM1 expression in CD133 + EpCAM + cells from Huh7, and HepG2 cell lines and HCC P1 patient sample. (B) RT-PCR analysis shows significant expression of stem cell factors in MACS-purified Huh7 CD133 + EpCAM + cells in comparison to CD133 - EpCAM - fractions (n=3 per group) (C) Flow cytometry dot plots depict reduction in CD133 + Epcam + LCSLCs during TREM1 silencing in Huh7 and HepG2 cells (n=4 per group). (D) RT-PCR analysis of MACS-purified LCSLCs reveals a significant decrease in stem cell factor expression during TREM1 ablation (n=3 per group). (E) Western blot analysis shows significant expression of stem cell proteins in MACS purified Huh7 and HepG2 control CD133 + EpCAM + cells in comparison to TREM1 KO CD133 + EpCAM + cells. (F) Spheroid formation assay shows TREM1 abrogation significantly limits spheroid formation and overall proliferation of LCSLCs. Keyence microscope was used for the acquisition of bright field images. Scale bars = 50 μm. Spheroids were counted using ImageJ (n=3 per group) (G) Colony formation assay demonstrates TREM1-positive LCSLCs form significantly more colonies than their KO counterparts. Representative images show TREM1 KO and Control Huh7 and HepG2 LCSLC colonies stained using crystal violet after 14 days. Data were plotted using GraphPad Prism (n=3 per group). (H) Representative images of tumors from NSG CDX models. 5000 Huh7 CD133 + EpCAM + LCSLCs from Control and TREM1 KO groups were injected subcutaneously (n=6 mice per group). The experiment was independently repeated three times for statistical analysis. ***p<0.001, ****p<0.0001.

Article Snippet: Followed by blocking with SuperBlock blocking buffer (PI37580, Thermo Fisher Scientific) for 1h, membranes were incubated with primary antibodies - TREM1 monoclonal (FERMA548755, Invitrogen,1:1000), CD133 Rabbit polyclonal (A0219, Abclonal, 1:1000), Epcam Rabbit polyclonal (A23654, Abclonal, 1:1000), OCT4 Rabbit polyclonal (A7920, Abclonal, 1:1000), Nanog Rabbit polyclonal (A3232, Abclonal, 1:1000), Sox2 Rabbit polyclonal (A19118, Abclonal, 1:1000), β Actin (MAB8929SP, R&D Systems,1:1000), α tubulin (sc-53029, Santa Cruz Biotechnology, 1:1000) at 4°C overnight.

Techniques: Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Purification, Comparison, Western Blot, Control, Tube Formation Assay, Microscopy, Colony Assay, Staining, Injection

Journal: Frontiers in Immunology

Article Title: TREM1 is essential for maintaining stemness of liver cancer stem-like cells in hepatocellular carcinoma

doi: 10.3389/fimmu.2025.1618342

Figure Lengend Snippet: Bulk RNA sequencing and pathway analysis of CD133 + EpCAM + cells from Huh7 control and TREM1 KO cell lines. (A) Heat map created using K-means clustering of the top 10,000 differentially expressed genes (DEGs) reveals that TREM1 KO in Huh7 LCSLCs significantly impacts cell proliferation pathways. Each sample was analyzed in triplicate. (B) Pathway analysis using Gene Set Enrichment Analysis (GSEA). The lollipop plot highlights the downregulated Gene Ontology (GO) Biological Process pathways in TREM1 KO Huh7 LCSLCs. (C) The lollipop plot shows the GO cellular components inhibited by the TREM1 KO, emphasizing the nuclear structures and complexes impacted. (D) Volcano plot displays the DEGs between Huh7 Ctrl and Huh7 TREM1 KO CD133 + EpCAM + cells. Genes with a log2 fold change > 1 and adjusted p- value < 0.05 are highlighted in red, indicating significant upregulation, while those with a log2 fold change < -1 and adjusted p -value < 0.05 are highlighted in blue, indicating significant downregulation. This plot underscores the downregulation of TREM1 and cancer stem cell-associated genes in the Huh7 CD133 + EpCAM + TREM1 KO cells.

Article Snippet: Followed by blocking with SuperBlock blocking buffer (PI37580, Thermo Fisher Scientific) for 1h, membranes were incubated with primary antibodies - TREM1 monoclonal (FERMA548755, Invitrogen,1:1000), CD133 Rabbit polyclonal (A0219, Abclonal, 1:1000), Epcam Rabbit polyclonal (A23654, Abclonal, 1:1000), OCT4 Rabbit polyclonal (A7920, Abclonal, 1:1000), Nanog Rabbit polyclonal (A3232, Abclonal, 1:1000), Sox2 Rabbit polyclonal (A19118, Abclonal, 1:1000), β Actin (MAB8929SP, R&D Systems,1:1000), α tubulin (sc-53029, Santa Cruz Biotechnology, 1:1000) at 4°C overnight.

Techniques: RNA Sequencing, Control

Journal: Frontiers in Immunology

Article Title: TREM1 is essential for maintaining stemness of liver cancer stem-like cells in hepatocellular carcinoma

doi: 10.3389/fimmu.2025.1618342

Figure Lengend Snippet: Bulk RNA sequencing and pathway analysis of CD133 + EpCAM + cells from HepG2 control and TREM1 KO cell lines. (A) Heat map created using K-means clustering of the top 10,000 differentially expressed genes (DEGs) reveals that TREM1 KO in HepG2 LCSLCs predominantly affects extracellular pathways. Each sample was analyzed in triplicate. (B) Pathway analysis using GSEA. The lollipop plot highlights the downregulated pathways in TREM1 KO HepG2 LCSLCs, focusing on significant extracellular pathways affected by the knockout. (C) The lollipop plot shows the GO cellular components inhibited by the TREM1 KO, emphasizing the extracellular structures and complexes impacted. (D) Volcano plot displays the DEGs between HepG2 Control and HepG2 KO CD133 + EpCAM + cells. Genes with a log2 fold change > 1 and adjusted p -value < 0.05 are highlighted in red, indicating significant upregulation, while those with a log2 fold change < -1 and adjusted p -value < 0.05 are highlighted in blue, indicating significant downregulation. This plot highlights the downregulation of TREM1 and cancer stem cell-associated genes in the HepG2 CD133 + EpCAM + TREM1 KO cells.

Article Snippet: Followed by blocking with SuperBlock blocking buffer (PI37580, Thermo Fisher Scientific) for 1h, membranes were incubated with primary antibodies - TREM1 monoclonal (FERMA548755, Invitrogen,1:1000), CD133 Rabbit polyclonal (A0219, Abclonal, 1:1000), Epcam Rabbit polyclonal (A23654, Abclonal, 1:1000), OCT4 Rabbit polyclonal (A7920, Abclonal, 1:1000), Nanog Rabbit polyclonal (A3232, Abclonal, 1:1000), Sox2 Rabbit polyclonal (A19118, Abclonal, 1:1000), β Actin (MAB8929SP, R&D Systems,1:1000), α tubulin (sc-53029, Santa Cruz Biotechnology, 1:1000) at 4°C overnight.

Techniques: RNA Sequencing, Control, Knock-Out

Journal: Frontiers in Immunology

Article Title: TREM1 is essential for maintaining stemness of liver cancer stem-like cells in hepatocellular carcinoma

doi: 10.3389/fimmu.2025.1618342

Figure Lengend Snippet: TREM1 inhibition via VJDT depletes LCSLCs, reduces tumor size, and decreases spheroid formation. (A) Tumor growth curves for Huh7 vehicle and VJDT treated mice (mean ± SEM, n=5 mice/group). Representative images of tumors from indicated groups on day 22. (B) Flow cytometry analysis shows a significant reduction in CD133 + EpCAM + LCSLCs in VJDT-treated tumors compared to the vehicle (n=4 per group). (C) Western blot analysis of two vehicle-treated and two VJDT-treated tumors shows reduced expression of stem cell-related proteins in VJDT-treated tumors. (D) Representative images from the spheroid formation assay demonstrate reduced spheroid formation following VJDT treatment. Scale bar = 50 µm. Spheroids were counted using ImageJ. **p<0.01, ***p<0.001, ns-not significant.

Article Snippet: Followed by blocking with SuperBlock blocking buffer (PI37580, Thermo Fisher Scientific) for 1h, membranes were incubated with primary antibodies - TREM1 monoclonal (FERMA548755, Invitrogen,1:1000), CD133 Rabbit polyclonal (A0219, Abclonal, 1:1000), Epcam Rabbit polyclonal (A23654, Abclonal, 1:1000), OCT4 Rabbit polyclonal (A7920, Abclonal, 1:1000), Nanog Rabbit polyclonal (A3232, Abclonal, 1:1000), Sox2 Rabbit polyclonal (A19118, Abclonal, 1:1000), β Actin (MAB8929SP, R&D Systems,1:1000), α tubulin (sc-53029, Santa Cruz Biotechnology, 1:1000) at 4°C overnight.

Techniques: Inhibition, Flow Cytometry, Western Blot, Expressing, Tube Formation Assay

Journal: Cell Death & Disease

Article Title: Temozolomide promotes glioblastoma stemness expression through senescence-associated reprogramming via HIF1α/HIF2α regulation

doi: 10.1038/s41419-025-07617-w

Figure Lengend Snippet: A Immunofluorescence analysis showed high expression levels of stemness markers CD133, Nestin, and transcription factors Sox2 and Klf4 in aggregation cells. B Elevated expression levels of CD133, Nestin, Sox2, and Klf4 were observed after two weeks of culturing aggregation cells. C Western blot analysis confirmed increased expression of stemness markers CD133, CD15, Nestin, Sox2, and Klf4 after one to two weeks of TMZ treatment, with higher expression levels in aggregation cells. D Aggregation cells (group ②) demonstrated greater invasive capacity compared to controls (group ①). Co-culturing control and aggregation cells showed a higher number of migrating cells when control cells were co-cultured with aggregation cells (group ④) than with control cells (group ③). E RFP-labeled aggregation cells co-cultured with aggregation cells exhibited the highest invasive capacity (group ④) compared to other groups, in the order: group ④ > group ③ (RFP-aggregation cells co-cultured with control cells) > group ② (RFP-control cells co-cultured with aggregation cells) > group ① (RFP-control cells co-cultured with control cells). * P < 0.05 was determined using Student’s t -test.

Article Snippet: These cells were incubated with polyclonal rabbit anti-human CD133 + IgGs (Miltenyi Biotech, Germany) at 4 °C for 15 min, washed with PBS containing 1% BSA, and resuspended in PBSE (108 cells/300 μl).

Techniques: Immunofluorescence, Expressing, Western Blot, Control, Cell Culture, Labeling

Journal: Cell Death & Disease

Article Title: Temozolomide promotes glioblastoma stemness expression through senescence-associated reprogramming via HIF1α/HIF2α regulation

doi: 10.1038/s41419-025-07617-w

Figure Lengend Snippet: A GSEA analysis indicated upregulation of senescence-associated hallmarks and inhibition of growth-promoting pathways. B The heatmap of U87 overlapping differentially expressed genes (DEGs) with SASP at the mRNA level revealed upregulation of most DEGs, including IL6, IL7, CXCL3, CXCL2, ICAM1, CCL2, CCL3, MMP7, and TIMP1. Conversely, senescence-inhibiting genes such as CDC25B, CDC25C, CDC25A, CDKN2D, MSH6, and MSH5 were downregulated. C RT-qPCR analysis demonstrated significant time-dependent increases in the expression of senescence-promoting genes, including IL1a, IL1b, IL6, IL8, CCL2, CDKN1A, CDKN2B, P53, and CXCL3, while the expression of the senescence-inhibiting gene MSH2 decreased significantly. D Mass spectrometry revealed high expression of senescence-promoting SASP proteins, including ITGA4, MMP15, FN1, IGFB3, and FAS, with a concomitant decrease in senescence-suppressing SASP proteins, MSH2 and MSH6. E ELISA confirmed increased expression of IL1a, IL6, and IL8 in a time-dependent manner following TMZ treatment. F SA-β-Gal staining revealed a significant time-dependent increase in β-Gal-positive cells, and the rate of β-Gal positivity was lower in aggregation cells compared to control cells under TMZ treatment. G C 12 FDG expression increased approximately threefold after one week of TMZ treatment in CD133 − CD15 − GBM cells, with lower levels of expression in aggregation cells under equivalent TMZ concentrations. H C 12 FDG-negative and -positive cells were cultured under TMZ for 21 days, showing significantly higher levels of CD133 and CD15 in C 12 FDG-positive cells over time, while CD133 and CD15 expression was not significant difference in C 12 FDG-negative cells. * P < 0.05 and # P > 0.05 were determined using Student’s t -test.

Article Snippet: These cells were incubated with polyclonal rabbit anti-human CD133 + IgGs (Miltenyi Biotech, Germany) at 4 °C for 15 min, washed with PBS containing 1% BSA, and resuspended in PBSE (108 cells/300 μl).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Staining, Control, Cell Culture

Journal: Cell Death & Disease

Article Title: Temozolomide promotes glioblastoma stemness expression through senescence-associated reprogramming via HIF1α/HIF2α regulation

doi: 10.1038/s41419-025-07617-w

Figure Lengend Snippet: A GSEA analysis demonstrated significant upregulation of hypoxia hallmark pathways in newly formed aggregation cells. B HIF1α and HIF2α knockout CD133 − CD15 − cells cultured under TMZ for two months exhibited the lowest expression levels of stemness markers CD133, CD15, Nestin, and transcription factors Sox2 and Klf4. Cells with simultaneous knockout showed the least expression, followed by single knockouts, compared to the control. C HIF1α and HIF2α knockout CD133 − CD15 − cells cultured with TMZ for two months exhibited almost no aggregation formation. Aggregation formation was significantly reduced in single knockouts compared to the control. D Apoptosis and necrosis rates were most significantly increased in cells with simultaneous HIF1α and HIF2α knockouts, followed by single knockouts, compared to the control. E Cell cycle analysis revealed that control cells arrested in the G2/M phase, while HIF1α and HIF2α knockout cells entered the S phase. F The rates of β-Gal-positive and C 12 FDG-positive cells decreased most significantly in simultaneous HIF1α and HIF2α knockout cells, with intermediate decreases in single knockouts compared to the control. G RT-qPCR analysis showed the lowest expression of SASP factors, including IL1a, IL1b, IL6, IL8, CCL2, and others, in simultaneous knockouts, followed by single knockouts, compared to the control in U118 CD133 − CD15 − cells. H GO analysis of miRNA sequences from simultaneous and single HIF1α and HIF2α knockouts revealed activation of terms associated with senescence and stemness, such as stem cell population maintenance, DNA replication, cell cycle arrest, centrosomes, and p53 binding. I KEGG pathway analysis of miRNA sequences from simultaneous and single HIF1α and HIF2α knockouts indicated activation of pathways associated with senescence and stemness, including cell cycle regulation, cellular senescence, Wnt signaling, regulation of pluripotency in stem cells, focal adhesion, and autophagy. * P < 0.05, ** P < 0.01, and # P > 0.05 were determined using Student’s t -test.

Article Snippet: These cells were incubated with polyclonal rabbit anti-human CD133 + IgGs (Miltenyi Biotech, Germany) at 4 °C for 15 min, washed with PBS containing 1% BSA, and resuspended in PBSE (108 cells/300 μl).

Techniques: Knock-Out, Cell Culture, Expressing, Control, Cell Cycle Assay, Quantitative RT-PCR, Activation Assay, Binding Assay

Journal: The Journal of Physiological Sciences : JPS

Article Title: Changes in adrenoceptor expression level contribute to the cellular plasticity of glioblastoma cells

doi: 10.1016/j.jphyss.2025.100016

Figure Lengend Snippet: The antibodies used for Western blotting.

Article Snippet: CD133 rabbit polyclonal , 1:2000 , Proteintech , 18470 −1-AP.

Techniques: Western Blot, Produced