Journal: BioMed Research International

Article Title: Establishment and Characterization of a Highly Metastatic Ovarian Cancer Cell Line

doi: 10.1155/2018/3972534

Figure Lengend Snippet: The percentage of cell populations with expressions of cell surface markers CD34, CD24, CD133, CD117, and CD44 in A2780-M and A2780 cells.

Article Snippet: Anti-CD117 (Cat: 11996-R007-PE) and anti-CD44 (Cat: 12211-MM02-FITC) antibodies were from Sino Biological Inc. (Beijing, China).

Techniques:

Journal: Cancers

Article Title: MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells

doi: 10.3390/cancers13112550

Figure Lengend Snippet: MARCH8 interacts with and degrades CD44 through the lysosome pathway. ( A ) Immunoblots to detect decreased CD44 and increased BAX, BID, and MARCH8 expression levels in MARCH8-overexpressing cells compared to GFP control cells. ( B ) Immunofluorescence staining with anti-CD44 antibody showing decreased expression of membrane protein CD44 (blue) in MARCH8-GFP expressing cells. ( C ) Flow cytometry histogram overlay (left panel) and dot plots (right panels) indicating MARCH8-decreased CD44 expression levels in negative association with MARCH8-GFP signals. ( D ) Immunoblots of endogenous CD44 and exogenous MARCH8-GFP after transient transfection of MARCH-GFP and treatment with MG-132 or chloroquine (CLQ) to block the proteasomal or lysosomal degradation pathways, respectively. ( E ) Immunoblots of MARCH8 and FLAG-tagged CD44 after anti-FLAG mediated immunoprecipitation (IP) of the lysates of HEK-293 cells after transfections with MARCH8-GFP and CD44-FLAG (standard isoform CD44s and full-length CD44f) and treatment with CLQ, indicating the interactions between MARCH8 and CD44 (CD44s or CD44f). ( F ) Immunoblots of BAX, BID, CD44, and MARCH8 in the MDA-MB-231 cells with stable expression of GFP or MARCH8-GFP with transient transfection of a FLAG vector control or restoration of CD44 expression via CD44s-FLAG. CD44 overexpression slightly inhibited the expression of proapoptotic BID and BAX in MARCH8-GFP-overexpressing cells.

Article Snippet: Plasmids that were used for overexpression include human CD44 standard form (NM_001001391), FLAG-tagged ORF Clone pCMV6-Flag-CD44s (OriGene Technologies, Rockville, MD, USA, Cat RC221820), CD44 full-length pCMV3-CD44f-HA ((Sino Biological, Beijing, China, Cat HG12211-CY), Lenti ORF clone of human MARCH8-GFP (OriGene Technologies, Rockville, MD, USA, Cat RC209891L2), GFP control (OriGene Technologies, Rockville, MD, USA, Cat PS10007), STAT3 (Addgene, Watertown, MA, USA, Cat 71450), and STAT3 Y705F (Addgene, Watertown, MA, USA, Cat 71445) mutant.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, Transfection, Blocking Assay, Immunoprecipitation, Plasmid Preparation, Over Expression

Journal: Cancers

Article Title: MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells

doi: 10.3390/cancers13112550

Figure Lengend Snippet: MARCH8 interacts with and degrades STAT3 through the proteasome pathway. ( A ) Immunoblots of AKT, pAKT, ERK, and pERK in MDA-MB-231 cells with stable expression of GFP and MARCH8-GFP, both adherent and in suspension. ( B ) Immunoblots of STAT3, pSTAT3 (Y705), CD44, and MARCH8 in GFP- and MARCH8-GFP-expressing cells in the absence or presence of MG-132 and CLQ, both adherent and in suspension. ( C ) Immunoblots of MARCH8, STAT3, pSTAT3 (Y705), and ubiquitin in lysates of cells co-transfected with STAT3 (wildtype or Y705F) and MARCH8-GFP (or GFP control) and immunoprecipitated by anti-STAT3 and anti-pSTAT3 (Y705). ( D ) Immunoblots of CD44, STAT3, BID, and BAX in MDA-MB-231 cells with stable expression of GFP and MARCH8-GFP, with transient transfection with CD44, STAT3, or mutant Y705F as indicated, both adherent and in suspension.

Article Snippet: Plasmids that were used for overexpression include human CD44 standard form (NM_001001391), FLAG-tagged ORF Clone pCMV6-Flag-CD44s (OriGene Technologies, Rockville, MD, USA, Cat RC221820), CD44 full-length pCMV3-CD44f-HA ((Sino Biological, Beijing, China, Cat HG12211-CY), Lenti ORF clone of human MARCH8-GFP (OriGene Technologies, Rockville, MD, USA, Cat RC209891L2), GFP control (OriGene Technologies, Rockville, MD, USA, Cat PS10007), STAT3 (Addgene, Watertown, MA, USA, Cat 71450), and STAT3 Y705F (Addgene, Watertown, MA, USA, Cat 71445) mutant.

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Mutagenesis

Journal: Molecular Medicine Reports

Article Title: Overexpression of TG2 enhances the differentiation of ectomesenchymal stem cells into neuron-like cells and promotes functional recovery in adult rats following spinal cord injury

doi: 10.3892/mmr.2019.10502

Figure Lengend Snippet: Identification of EMSCs. (A) A representative image of primary EMSCs. Phase-contract images of EMSCs from passages (B) 3 and (C) 5. EMSCs of passage 3 positively expressed neural crest cell and mesenchymal stem cell markers, including (D) vimentin, (E) s100, (F) CD44 and (G) nestin. Scale bar, 50 µm. EMSCs, ectomesenchymal stem cells.

Article Snippet: EMSCs (passage 3) were identified by detecting the expression of nestin (rabbit polyclonal, ab27952, Abcam), vimentin (mouse monoclonal; cat. no. BM0135; Wuhan Boster Biological Technology, Ltd.; 1:400), CD133 (rabbit polyclonal; cat. no. BA3993-2; Wuhan Boster Biological Technology, Ltd.; 1:400) and CD44 (rabbit polyclonal; cat. no. A00052; Wuhan Boster Biological Technology, Ltd.; 1:400) by immunofluorescence staining according to the protocol described in the following section.

Techniques:

Journal: Oncology Letters

Article Title: OTUD7B knockdown inhibits the proliferation and stemness of breast cancer cells by destabilizing FOXM1

doi: 10.3892/ol.2024.14235

Figure Lengend Snippet: OTUD7B knockdown reduces the stemness and proliferation of MDA-MB-468 cells. (A) Western blotting of SOX2 and Nanog proteins in OTUD7B-knockdown MDA-MB-468 cells. (B) Flow cytometric analysis of CD44 and EpCAM proteins in OTUD7B-knockdown MDA-MB-468 cells. (C) Statistical analysis of mean fluorescence intensity. (D) Tumorsphere diameter analysis of OTUD7B-knockdown MDA-MB-468 cells. (E) Cell Counting Kit 8 analysis of cell proliferation in OTUD7B-knockdown MDA-MB-468 cells. (F) Colony formation capacity analysis of OTUD7B-knockdown MDA-MB-468 cells. **P<0.01, ***P<0.001 and ****P<0.0001. OTUD7B, OTU domain-containing 7B; NC, negative control; sh, short hairpin; MFI, mean fluorescence intensity.

Article Snippet: After 24 h, the cells were harvested and incubated with FITC-conjugated antibodies for CD44 (cat. no. 12211-MM02-F; 1:200; Sino Biological, Inc.), CD24 (cat. no. 655154; 1:200; BD Biosciences) or EpCAM (cat. no. 10694-MM06-F; 1:200; Sino Biological, Inc.) at 26°C for 30 min.

Techniques: Western Blot, Fluorescence, Cell Counting, Negative Control

Journal: Oncology Letters

Article Title: OTUD7B knockdown inhibits the proliferation and stemness of breast cancer cells by destabilizing FOXM1

doi: 10.3892/ol.2024.14235

Figure Lengend Snippet: Overexpression of FOXM1 increases the expression levels of stemness markers in OTUD7B-knockdown breast cancer cells. (A) Flow cytometric analysis of the expression levels of CD44, CD24 and EpCAM in MDA-MB-468, MDA-MB-453 and MCF7 cells. (B) Statistical analysis of the MFI of CD44, CD24 and EpCAM in MDA-MB-468, MDA-MB-453 and MCF7 cells. **P<0.01, ***P<0.001 and ****P<0.0001. OTUD7B, OTU domain-containing 7B; FOXM1, forkhead box protein M1; NC, negative control; sh, short hairpin; MFI, mean fluorescence intensity.

Article Snippet: After 24 h, the cells were harvested and incubated with FITC-conjugated antibodies for CD44 (cat. no. 12211-MM02-F; 1:200; Sino Biological, Inc.), CD24 (cat. no. 655154; 1:200; BD Biosciences) or EpCAM (cat. no. 10694-MM06-F; 1:200; Sino Biological, Inc.) at 26°C for 30 min.

Techniques: Over Expression, Expressing, Negative Control, Fluorescence

Journal: Cell reports

Article Title: BIRC2 Expression Impairs Anti-Cancer Immunity and Immunotherapy Efficacy.

doi: 10.1016/j.celrep.2020.108073

Figure Lengend Snippet: Figure 4. BIRC2 Knockdown in Melanoma Cells Decreases Tumor Growth and Alters Inflammatory Cell Recruitment to the Tumor Micro- environment (A) B16F10 subclones expressing NTC or BIRC2 shRNA (sh3 or sh4) were implanted subcutaneously in female C57BL/6 mice, and tumor growth was monitored. (B–F) Tumors were harvested on day 35 and the percentage of CD8+/CD44+/CD69+ activated T cells (B), CD11b+/NK1.1+ NK cells (C), CD11b+/CD11c+/F4/80

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse BIRC2 Novus Biologicals Cat# NB100-56889 Anti-mouse b-Actin Santa Cruz Biotechnology Cat# sc-47778 Anti-mouse CD3 BioLegend Cat# 102102 Anti-mouse CD3 Novus Biologicals Cat# FAB4841G-100 Anti-mouse CD4 Novus Biologicals Cat# FAB554A-100 Anti-mouse CD8A Novus Biologicals Cat# NBP1-49045PE Anti-mouse CD11b Novus Biologicals Cat# NB110-89474AF405 Anti-mouse CD11c Novus Biologicals Cat# NB110-40766AF488 Anti-mouse CD25 Novus Biologicals Cat# NBP2-27425AF488 Anti-mouse CD28 BioLegend Cat# 100223 Anti-mouse CD44 Novus Biologicals Cat# NBP1-47386APC Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF488 Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF405 Anti-mouse CD69 Novus Biologicals Cat# NBP1-28011AF488 Anti-mouse CD80 Novus Biologicals Cat# NBP1-43385AF488 Anti-mouse CD314 Novus Biologicals Cat# FAB1547V-100UG Anti-mouse F4/80 Novus Biologicals Cat# NB600-404APC Anti-mouse FoxP3 Novus Biologicals Cat# NB100-39002PE Anti-human HIF-1a Novus Biologicals Cat# NB100-479 Anti-human HIF-1b Novus Biologicals Cat# NB100-124 Anti-human HIF-2a Novus Biologicals Cat# NB100-122 Anti-mouse IFNG Novus Biologicals Cat# IC485V-100UG Anti-mouse Ly6c Novus Biologicals Cat# NBP1-28046AF488 Anti-mouse Ly6g Novus Biologicals Cat# FAB1037A-100 Anti-mouse NK1.1 Novus Biologicals Cat# NB100-77528APC Anti-mouse p50 Novus Biologicals Cat# NBP2-6735 Anti-mouse Rel A Novus Biologicals Cat# NB100-2176 Anti-mouse Rel B Novus Biologicals Cat# NBP2-20123 Anti-mouse a-Tubulin Novus Biologicals Cat# NB600-506 Armenian Hamster IgG, anti-mouse CXCL9 (MIG) Bio X Cell Cat# BE0309 Polyclonal Armenian hamster IgG Bio X Cell Cat# BE0091 Syrian Hamster IgG, anti-mouse CTLA-4 Bio X Cell Cat# BP0131 Rat IgG2a, k, anti-mouse PD-1 (CD279) Bio X Cell Cat# BP0146 Rat IgG2a isotype control Bio X Cell Cat# BE0089 Chemicals, Peptides, and Recombinant Proteins Acriflavine Sigma Aldrich SKU # A8126 TRIzol Reagent Invitrogen Cat# 15596026 Puromycin Dihydrochloride ThermoFisher Cat# A1113803 ECL Prime Western Blotting System GE Healthcare SKU# GERPN2232 PolyJet In Vitro DNA Transfection Reagent Signagen Cat # SL100688 Rabbit anti-mouse IgG-HRP Santa Cruz Biotech Cat# sc-358914 Rabbit IgG HRP Linked Whole Ab GE Healthcare SKU# GENA934 (Continued on next page) e1 Cell Reports 32, 108073, August 25, 2020

Techniques: Knockdown, Expressing, shRNA

Journal: Cell reports

Article Title: BIRC2 Expression Impairs Anti-Cancer Immunity and Immunotherapy Efficacy.

doi: 10.1016/j.celrep.2020.108073

Figure Lengend Snippet: Figure 5. BIRC2 Knockdown in Breast Can- cer Cells Decreases Tumor Growth and Al- ters Inflammatory Cell Recruitment to the Tumor Microenvironment (A) EMT6 subclones expressing NTC or either of two shRNAs targeting BIRC2 (sh4 and sh5) were cultured at 20% O2 and analyzed for expression of BIRC2 protein by immunoblot assay. (B) EMT6 subclones (NTC, sh4, and sh5) were im- planted into the mammary fat pad of female BALB/c mice, and tumor volumes were determined (mean ± SEM; n = 4); *p < 0.05 (Kruskal-Wallis test with Benjamini-Hochberg post-test). (C–F) Tumors were harvested on day 13, and the percentage of CD8+/CD44+/CD69+ activated T cells (C), CD3/NK1.1+ NK cells (D), CD11b+/F4/ 80/CD11c+ DCs (E), and CD11b+/Ly6C+ MDSCs (F) was determined (mean ± SEM; n = 4); *p < 0.05 for the indicated pairs (Kruskal-Wallis test with Benjamini-Hochberg post-test). All immune cell populations were calculated as a percentage of the total number of live cells (based on forward and side scatter). (G) EMT6 subclones were implanted into the mammary fat pad of female SCID mice, and tumor growth was monitored. See also Figure S3B.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse BIRC2 Novus Biologicals Cat# NB100-56889 Anti-mouse b-Actin Santa Cruz Biotechnology Cat# sc-47778 Anti-mouse CD3 BioLegend Cat# 102102 Anti-mouse CD3 Novus Biologicals Cat# FAB4841G-100 Anti-mouse CD4 Novus Biologicals Cat# FAB554A-100 Anti-mouse CD8A Novus Biologicals Cat# NBP1-49045PE Anti-mouse CD11b Novus Biologicals Cat# NB110-89474AF405 Anti-mouse CD11c Novus Biologicals Cat# NB110-40766AF488 Anti-mouse CD25 Novus Biologicals Cat# NBP2-27425AF488 Anti-mouse CD28 BioLegend Cat# 100223 Anti-mouse CD44 Novus Biologicals Cat# NBP1-47386APC Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF488 Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF405 Anti-mouse CD69 Novus Biologicals Cat# NBP1-28011AF488 Anti-mouse CD80 Novus Biologicals Cat# NBP1-43385AF488 Anti-mouse CD314 Novus Biologicals Cat# FAB1547V-100UG Anti-mouse F4/80 Novus Biologicals Cat# NB600-404APC Anti-mouse FoxP3 Novus Biologicals Cat# NB100-39002PE Anti-human HIF-1a Novus Biologicals Cat# NB100-479 Anti-human HIF-1b Novus Biologicals Cat# NB100-124 Anti-human HIF-2a Novus Biologicals Cat# NB100-122 Anti-mouse IFNG Novus Biologicals Cat# IC485V-100UG Anti-mouse Ly6c Novus Biologicals Cat# NBP1-28046AF488 Anti-mouse Ly6g Novus Biologicals Cat# FAB1037A-100 Anti-mouse NK1.1 Novus Biologicals Cat# NB100-77528APC Anti-mouse p50 Novus Biologicals Cat# NBP2-6735 Anti-mouse Rel A Novus Biologicals Cat# NB100-2176 Anti-mouse Rel B Novus Biologicals Cat# NBP2-20123 Anti-mouse a-Tubulin Novus Biologicals Cat# NB600-506 Armenian Hamster IgG, anti-mouse CXCL9 (MIG) Bio X Cell Cat# BE0309 Polyclonal Armenian hamster IgG Bio X Cell Cat# BE0091 Syrian Hamster IgG, anti-mouse CTLA-4 Bio X Cell Cat# BP0131 Rat IgG2a, k, anti-mouse PD-1 (CD279) Bio X Cell Cat# BP0146 Rat IgG2a isotype control Bio X Cell Cat# BE0089 Chemicals, Peptides, and Recombinant Proteins Acriflavine Sigma Aldrich SKU # A8126 TRIzol Reagent Invitrogen Cat# 15596026 Puromycin Dihydrochloride ThermoFisher Cat# A1113803 ECL Prime Western Blotting System GE Healthcare SKU# GERPN2232 PolyJet In Vitro DNA Transfection Reagent Signagen Cat # SL100688 Rabbit anti-mouse IgG-HRP Santa Cruz Biotech Cat# sc-358914 Rabbit IgG HRP Linked Whole Ab GE Healthcare SKU# GENA934 (Continued on next page) e1 Cell Reports 32, 108073, August 25, 2020

Techniques: Knockdown, Expressing, Cell Culture, Western Blot

Journal: Cell reports

Article Title: BIRC2 Expression Impairs Anti-Cancer Immunity and Immunotherapy Efficacy.

doi: 10.1016/j.celrep.2020.108073

Figure Lengend Snippet: Figure 6. BIRC2 Knockdown in B16F10 Cells Increases Anti-tumor Immunity by Increasing CXCL9 Expression (A) NTC and BIRC2-KD subclones were implanted into C57BL/6 mice. When BIRC2-KD tumors became palpable, mice were treated with anti-CXCL9 or IgG every 3 days. Tumor volumes were determined (mean ± SEM; n = 4); *p < 0.05 (Kruskal-Wallis test with Benjamini-Hochberg post-test). (B–E) Tumors were harvested on day 35, and the percentage of CD8+ T cells (relative to CD45+ population) (B), CD8+/CD44+/CD69+ T cells (C), CD3/NK1.1+ NK cells (D), and CD11b+/CD11c+/F4/80 DCs (E) was determined (mean ± SEM; n = 4); *p < 0.05 (Kruskal-Wallis test with Benjamini-Hochberg post-test). All immune cell populations (except B) were calculated as a percentage of the total live cells (based on forward and side scatter). (F–H) The Pearson correlation test was performed to compare CXCL9 mRNA expression with CD8+ T cell score (F), NK cell score (G), and DC score (H), using TCGA data from 481 human melanomas. See also Figures S3C and S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse BIRC2 Novus Biologicals Cat# NB100-56889 Anti-mouse b-Actin Santa Cruz Biotechnology Cat# sc-47778 Anti-mouse CD3 BioLegend Cat# 102102 Anti-mouse CD3 Novus Biologicals Cat# FAB4841G-100 Anti-mouse CD4 Novus Biologicals Cat# FAB554A-100 Anti-mouse CD8A Novus Biologicals Cat# NBP1-49045PE Anti-mouse CD11b Novus Biologicals Cat# NB110-89474AF405 Anti-mouse CD11c Novus Biologicals Cat# NB110-40766AF488 Anti-mouse CD25 Novus Biologicals Cat# NBP2-27425AF488 Anti-mouse CD28 BioLegend Cat# 100223 Anti-mouse CD44 Novus Biologicals Cat# NBP1-47386APC Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF488 Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF405 Anti-mouse CD69 Novus Biologicals Cat# NBP1-28011AF488 Anti-mouse CD80 Novus Biologicals Cat# NBP1-43385AF488 Anti-mouse CD314 Novus Biologicals Cat# FAB1547V-100UG Anti-mouse F4/80 Novus Biologicals Cat# NB600-404APC Anti-mouse FoxP3 Novus Biologicals Cat# NB100-39002PE Anti-human HIF-1a Novus Biologicals Cat# NB100-479 Anti-human HIF-1b Novus Biologicals Cat# NB100-124 Anti-human HIF-2a Novus Biologicals Cat# NB100-122 Anti-mouse IFNG Novus Biologicals Cat# IC485V-100UG Anti-mouse Ly6c Novus Biologicals Cat# NBP1-28046AF488 Anti-mouse Ly6g Novus Biologicals Cat# FAB1037A-100 Anti-mouse NK1.1 Novus Biologicals Cat# NB100-77528APC Anti-mouse p50 Novus Biologicals Cat# NBP2-6735 Anti-mouse Rel A Novus Biologicals Cat# NB100-2176 Anti-mouse Rel B Novus Biologicals Cat# NBP2-20123 Anti-mouse a-Tubulin Novus Biologicals Cat# NB600-506 Armenian Hamster IgG, anti-mouse CXCL9 (MIG) Bio X Cell Cat# BE0309 Polyclonal Armenian hamster IgG Bio X Cell Cat# BE0091 Syrian Hamster IgG, anti-mouse CTLA-4 Bio X Cell Cat# BP0131 Rat IgG2a, k, anti-mouse PD-1 (CD279) Bio X Cell Cat# BP0146 Rat IgG2a isotype control Bio X Cell Cat# BE0089 Chemicals, Peptides, and Recombinant Proteins Acriflavine Sigma Aldrich SKU # A8126 TRIzol Reagent Invitrogen Cat# 15596026 Puromycin Dihydrochloride ThermoFisher Cat# A1113803 ECL Prime Western Blotting System GE Healthcare SKU# GERPN2232 PolyJet In Vitro DNA Transfection Reagent Signagen Cat # SL100688 Rabbit anti-mouse IgG-HRP Santa Cruz Biotech Cat# sc-358914 Rabbit IgG HRP Linked Whole Ab GE Healthcare SKU# GENA934 (Continued on next page) e1 Cell Reports 32, 108073, August 25, 2020

Techniques: Knockdown, Expressing

Journal: International Journal of Oncology

Article Title: PD-L1 mediates triple-negative breast cancer evolution via the regulation of TAM/M2 polarization

doi: 10.3892/ijo.2022.5440

Figure Lengend Snippet: αPD-L1 inhibits the EMT process and the stemness of TNBC promoted by TAM/M2. (A) Protein expression levels of EMT markers, E-Cadherin, ZO-1, N-Cadherin, Vimentin, Slug and Twist, in MDA-MB-231 and Hs578T cells treated with CM, as determined using western blot analysis. (B) E-cadherin and vimentin protein expression levels in MDA-MB-231 and Hs578T cells treated with CM were analyzed using IF staining. The markers were all expressed in the cytoplasm (magnification, ×400). (C) Protein expression levels of the stemness markers, CD44, Oct4, Nanog, Bmi1 and Sox2, in MDA-MB-231 and Hs578T cells treated with CM, as determined using western blot analysis. (D) CD44 protein expression levels in MDA-MB-231 and Hs578T cells treated with CM were assessed using IF staining. The markers were all expressed in the cytoplasm (magnification, ×400). * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001. αPD-L1, programmed death-ligand 1 inhibitor; EMT, epithelial-mesenchymal transition; TAM/M2, tumor-associated macrophages/M2-type; ZO-1, zonula occludens-1; CM, conditional media; IF, immunofluorescence.

Article Snippet: Primary antibodies specific for the following proteins were used: CD206 (1:100; cat. no. 24595; CST Biological Reagents Co., Ltd.), E-cadherin (1:100; cat. no. 14472; CST Biological Reagents Co., Ltd.), vimentin (1:100; cat. no. sc-6260; Santa Cruz Biotechnology, Inc.), CD44 (1:100; cat. no. 3570; CST Biological Reagents Co., Ltd.), VEGF (1:100; cat. no. sc-7269; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Western Blot, Staining, Immunofluorescence

Journal: International Journal of Oncology

Article Title: PD-L1 mediates triple-negative breast cancer evolution via the regulation of TAM/M2 polarization

doi: 10.3892/ijo.2022.5440

Figure Lengend Snippet: αPD-L1 reverses TNBC malignant evolution in the co-culture of TNBC cells and TAMs. (A and B) MDA-MB-231 and Hs578T cell migration capacity was assessed using wound healing and Transwell assays when the cells were treated with or without αPD-L1 in a co-culture system (magnification, ×200). (C) Protein expression levels of VEGF, MMP2 and MMP9 in MDA-MB-231 and Hs578T cells, with or without αPD-L1 in a co-culture system, were determined using western blot analysis. (D) Protein expression levels of the EMT markers, E-cadherin, ZO-1, N-cadherin, vimentin, Slug and Twist, in MDA-MB-231 and Hs578T cells, with or without αPD-L1 in a co-culture system, were determined using western blot analysis. (E) Protein expression levels of the stemness markers, CD44, Oct4, Nanog, Bmi1 and Sox2 in MDA-MB-231 and Hs578T cells, with or without αPD-L1 in a co-culture system were determined using western blot analysis. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001. αPD-L1, programmed death-ligand 1 inhibitor; TNBC, triple-negative breast cancer; TAM/M2, tumor-associated macrophages/M2-type; VEGF, vascular endothelial growth factor; EMT, epithelial-mesenchymal transition; ZO-1, zonula occludens-1.

Article Snippet: Primary antibodies specific for the following proteins were used: CD206 (1:100; cat. no. 24595; CST Biological Reagents Co., Ltd.), E-cadherin (1:100; cat. no. 14472; CST Biological Reagents Co., Ltd.), vimentin (1:100; cat. no. sc-6260; Santa Cruz Biotechnology, Inc.), CD44 (1:100; cat. no. 3570; CST Biological Reagents Co., Ltd.), VEGF (1:100; cat. no. sc-7269; Santa Cruz Biotechnology, Inc.).

Techniques: Co-Culture Assay, Migration, Expressing, Western Blot

Journal: International Journal of Oncology

Article Title: PD-L1 mediates triple-negative breast cancer evolution via the regulation of TAM/M2 polarization

doi: 10.3892/ijo.2022.5440

Figure Lengend Snippet: αPD-L1 inhibits lung metastasis in vivo . (A) Schematic diagram of the animal assays performed. (B and C) Tumor volume in the subcutaneous cell-transplanted mice was recorded over time throughout the animal assay. (D and H) The number of lung metastatic nodules was determined. (E) Lung metastases in mice that were subcutaneously injected with TNBC cells. Lung tissue images, H&E scan images and H&E magnification images (magnification, ×100) are shown, respectively. (F and G) Body weight of subcutaneous and intravenous cell-transplanted mice was recorded over time throughout the animal assay. (I) Lung metastases in mice that were intravenously injected with TNBC cells. Lung tissue images, H&E scan images and H&E magnification images (magnification, ×100) are shown, respectively. (J and K) CD206 protein expression levels in lung and tumor tissues were assessed using immunohistochemistry (magnification: upper panels, ×100, lower panels, ×200). (L-O) Protein expression levels of E-cadherin, vimentin, VEGF and CD44 in tumors tissues were determined using immunohistochemistry (magnification: upper panels, ×100, lower panels, ×200). * P<0.05. αPD-L1, programmed death-ligand 1 inhibitor; TNBC, triple-negative breast cancer; VEGF, vascular endothelial growth factor.

Article Snippet: Primary antibodies specific for the following proteins were used: CD206 (1:100; cat. no. 24595; CST Biological Reagents Co., Ltd.), E-cadherin (1:100; cat. no. 14472; CST Biological Reagents Co., Ltd.), vimentin (1:100; cat. no. sc-6260; Santa Cruz Biotechnology, Inc.), CD44 (1:100; cat. no. 3570; CST Biological Reagents Co., Ltd.), VEGF (1:100; cat. no. sc-7269; Santa Cruz Biotechnology, Inc.).

Techniques: In Vivo, Injection, Expressing, Immunohistochemistry