Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: Proteomic analysis of nuclear extracts from PD-L1 Cont (Sh-Cont) vs. PD-L1 KD (ShPD-L1(a) and Sh-PDL1(b)) clones of MDA-MB-231 breast cancer cells. Schematic overview of the proteomics-guided workflow used to identify PD-L1-dependent regulators of the SKP2/p21/p27 axis. The heatmap displays the top nuclear differentially expressed proteins (DEPs) in Sh-Cont compared to Sh-PD-L1(a) and Sh-PD-L1(b) (|log 2 FC| ≥ 2 and adjusted p -value ≤ 0.05). DEPs were further filtered based on literature-based relevance to proliferation, the PI3K/AKT pathway, and SKP2, p21, and/or p27 expression (details are in ).

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Clone Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: Knockdown of Livin and Galectin-1 downregulates SKP2 expression and inhibits colony formation. Expression levels of SKP2 ( A ), p21 ( B ), and p27 ( C ) in MDA-MB-231 cells following transient knockdown of six candidate upstream proteins measured using qIF. Data were normalized to negative control siRNA (si-Neg) and are presented as means ± SEMs from four independent experiments. ( D ) Representative IF images (×200 magnification) showing SKP2, p21, and p27 expression after Livin or Galectin-1 knockdown compared with control siRNA. ( E ) Colony-forming ability of MDA-MB-231 cells after transient knockdown of candidate genes. Data were normalized to negative control siRNA–treated cells and are displayed as means ± SEMs from three independent experiments ( n = 3). ( F ) Representative colony-forming assays following knockdown of PD-L1, Livin, Galectin-1, EIF1AX, CALM2, and TCF-3 compared to control siRNA. Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01.

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Knockdown, Expressing, Negative Control, Control

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: PD-L1 promotes Livin and Galectin-1 expression in TNBC cells. ( A ) Livin and Galectin-1 expression levels in PD-L1 KD MDA-MB-231 clones compared with PD-L1 Cont controls, measured using qIF. Data were normalized to Sh-Cont MFI and are presented as means ± SEMs from four independent experiments. ( B ) Representative IF images of PD-L1 KD clones and controls (×200 magnification). ( C ) Western blot analysis of Livin and Galectin-1 expression following PD-L1 knockdown in MDA-MB-231 cells. ( D ) Densitometric quantification of the Western blots from three independent experiments presented as mean ± SEM ( n = 3). Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01.

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Expressing, Clone Assay, Western Blot, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: PD-L1 overexpression in MDA-MB-468 breast cancer cells upregulates SKP2 in a Livin/Galectin-1-dependent manner. ( A ) PD-L1 expression measured by flow cytometry following stable overexpression of PD-L1 in MDA-MB-468 cells using a PD-L1 ORF construct (PD-L1-ORF), compared with empty vector-transfected cells (vector control). ( B ) Expression of Livin, Galectin-1, and SKP2 in stable PD-L1 ORF MDA-MB-468 cells, measured using qIF. ( C ) SKP2 expression upon transient knockdown of Livin, Galectin-1, or their combination (combined) in PD-L1-ORF and vector control MDA-MB-468 cells. Data were normalized to empty vector cells and are shown as means ± SEMs from three independent experiments (right) ( n = 3). Representative IF images (×200 magnification) (left). Statistical significance is indicated as follows: * p < 0.05, *** p < 0.001, NS, not significant.

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Over Expression, Expressing, Flow Cytometry, Construct, Plasmid Preparation, Transfection, Control, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: Knockdown of SKP2, Livin, or Galectin-1 abrogates PD-L1-promoted TNBC cell proliferation. MDA-MB-231 breast cancer cell proliferation was monitored in real time using the RTCA system. Cell index values are shown over 72 h ( upper panel , line graph) and at the 72 h endpoint ( lower panels , bar graphs; n = 3). Cells were transfected with siRNA targeting PD-L1 alone or in combination with Livin ( A ) or Galectin-1 ( B ), with or without SKP2 ( C , D ), and were compared with scrambled siRNA controls. Data are presented as mean ± SEM from at least three independent experiments ( n = 3). Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01; NS, not significant.

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Knockdown, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: PD-L1 modulates Livin and Galectin-1 expression in vivo. Expression of Livin and Galectin-1 in PD-L1 Cont and PD-L1 KD MDA-MB-231 xenografts. ( Left ) Representative IF images (×100 magnification). ( Right ) Bar graphs showing expression levels (mean ± SEM), measured using qIF, normalized to Sh-Cont from three different experiments ( n = 3). (*) indicates statistical significance ( p < 0.05).

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Expressing, In Vivo

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: PD-L1 upregulates Livin/Galectin-1 expression, activating the PI3K/AKT pathway to promote proliferation through SKP2–p21/p27. Schematic illustration of PD-L1-mediated PI3K/AKT activation via Livin and Galectin-1, which sustains PI3K/AKT signaling through a positive feedback loop. This PI3K/AKT activation upregulates SKP-2 and downregulates p21/p27 expression to drive cell cycle progression. Arrows indicate a promoting effect.

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Expressing, Activation Assay