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: Among the knocked-down candidates, Livin, EIF1AX, or Galectin-1 significantly reduced SKP2 expression as shown by quantitative immunofluorescence (qIF) ( A).

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: Among the knocked-down candidates, Livin, EIF1AX, or Galectin-1 significantly reduced SKP2 expression as shown by quantitative immunofluorescence (qIF) ( A).

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: Among the knocked-down candidates, Livin, EIF1AX, or Galectin-1 significantly reduced SKP2 expression as shown by quantitative immunofluorescence (qIF) ( A).

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: Among the knocked-down candidates, Livin, EIF1AX, or Galectin-1 significantly reduced SKP2 expression as shown by quantitative immunofluorescence (qIF) ( A).

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: Among the knocked-down candidates, Livin, EIF1AX, or Galectin-1 significantly reduced SKP2 expression as shown by quantitative immunofluorescence (qIF) ( A).

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: Livin and Galectin-1 promote PI3K/AKT pathway activation and vice versa. ( A ) Top: quantitative immunofluorescence (qIF) showing MFI of p-AKT (S473) in MDA-MB-231 cells upon transient knockdown of Livin, Galectin-1, or SKP2, normalized to si-Neg and presented as means ± SEMs from three different experiments. Bottom: representative IF images (×200 magnification). ( B ) Left: representative IF images (×200 magnification) showing Livin, Galectin-1, and SKP2 expression after PI3K inhibition with LY294002 treatment. Right: bar graph with normalized qIF data shown as means ± SEMs from three independent experiments ( n = 3). (*, # and NS) indicates statistical significance (* statistically significant p < 0.05, # = borderline significance, NS = not significant).

Article Snippet: Among the knocked-down candidates, Livin, EIF1AX, or Galectin-1 significantly reduced SKP2 expression as shown by quantitative immunofluorescence (qIF) ( A).

Techniques: Activation Assay, Immunofluorescence, Knockdown, Expressing, Inhibition

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: Among the knocked-down candidates, Livin, EIF1AX, or Galectin-1 significantly reduced SKP2 expression as shown by quantitative immunofluorescence (qIF) ( A).

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: Among the knocked-down candidates, Livin, EIF1AX, or Galectin-1 significantly reduced SKP2 expression as shown by quantitative immunofluorescence (qIF) ( A).

Techniques: Expressing, Activation Assay

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) Western blot analysis of Myc, Skp2, and p27 expression in human mesenchymal stem cells (hMSCs) and Ewing sarcoma cells (SKES1) treated with specific siRNAs or negative control siRNA. GAPDH was used as a loading control. The inclusion of hMSCs as a non-malignant comparator clarifies disease-specific expression differences. There were decreases in each mRNA by Myc-siRNA (20 nM) and (B) Skp2-siRNA (20 nM) (B) . The expression of Myc protein was analyzed by (C) immunoblotting and (D) densitometric quantification. The effect of Skp2-siRNA on Skp2 protein expression was analyzed by (E) immunoblotting and (F) quantified accordingly.

Article Snippet: Expression vectors: AP4 (12101), Skp2 (89142), and Myc (26818) were obtained from Addgene (Watertown, MA, USA).

Techniques: Western Blot, Expressing, Negative Control, Control

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) The cell growth of SKES and RDES cells was significantly decreased by Myc-KD. (B) Similarly, cell growth in both cell lines was suppressed by Skp2-KD. (C) The possibility that the decrease in cell growth was caused by apoptosis was evaluated; (D) Apoptosis was not induced under conditions where cell growth suppression was observed. The results are represented as the mean ± SEM (n.s., no significance). **, p < 0.01 versus the related untreated groups.

Article Snippet: Expression vectors: AP4 (12101), Skp2 (89142), and Myc (26818) were obtained from Addgene (Watertown, MA, USA).

Techniques:

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) The effect of Myc and Skp2 knockdown on the cell cycle was examined to determine whether growth suppression was due to cell cycle delay. In Myc-KD, Skp2-KD, and simultaneous Myc- and Skp2-KD groups, the S phase and G2/M phase populations decreased, while the G1 phase population increased. (B) These populations were quantified. The simultaneous knockdown group showed the most prominent changes. The results are represented as the mean ± SEM (n.s., no significance). **, p < 0.01 versus the related control groups.

Article Snippet: Expression vectors: AP4 (12101), Skp2 (89142), and Myc (26818) were obtained from Addgene (Watertown, MA, USA).

Techniques: Knockdown, Control

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) Tumor growth was compared across groups after tumor tissues were transplanted subcutaneously into mice. (B) The expression of p27 and CCNE in mouse tumor tissues was analyzed by immunohistochemistry. Original magnification, × 400; Scale bars: 50 μm. (C) The proportion of immunohistochemically positive cells was quantified. Data represent mean ± SEM of three independent experiments. **, p < 0.01 versus the related control groups. (D) A schematic summary of the study was created, demonstrating that Myc enhances the expression of AP4 and Skp2, ultimately leading to p27 ubiquitination.

Article Snippet: Expression vectors: AP4 (12101), Skp2 (89142), and Myc (26818) were obtained from Addgene (Watertown, MA, USA).

Techniques: Expressing, Immunohistochemistry, Control, Ubiquitin Proteomics

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: E3 ubiquitin ligase SKP2 limits autophagosome formation during Staphylococcus aureus infection

doi: 10.3389/fcimb.2026.1749151

Figure Lengend Snippet: Increased abundance of SKP2 upon S. aureus infection in A549 cells. A549 cells were infected with S. aureus USA300 (MOI 10) for the depicted time points. (A) SKP2 and IκBα abundance was analyzed by immunoblot compared to uninfected controls. (B, C) Densitometric analysis of SKP2 and IκBα normalized to β-actin and relative expression was calculated to the 2 h uninfected control, n = 3. (D) Analysis of nuclear and cytoplasmic fractionation for p65 abundance at 5 h post infection with S. aureus USA300 (MOI 10) with Lamin A serving as control for the nuclear fraction and MEK 1/2 serving as control for the cytoplasmic fraction, n = 3, one representative blot is shown . (E, F) Cytokine secretion was assessed via ELISA for IL-6, n = 3 (E) and IL-8, n = 3 (F) . (G) Cytotoxicity was monitored by determining extracellular LDH at 2 h, 3 h, and 5 h post-infection, n = 3. Data in (B–C) and (E–G) are presented as mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.001, students t-test).

Article Snippet: For immunoblotting, proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with primary antibodies against SKP2 (#2652, LOT:5), IκBα (#4814, LOT:21), LC3 (#12741, LOT:5), p27 (#3686, LOT:8), β-actin (#3700, LOT:21), lamin A (#86846, LOT:3), MEK1/2 (#4694, LOT:2; all from Cell Signaling Technology), anti-acetyl lysine antibody (#ab21623, LOT: 1010926-6; Abcam) and subsequently detected using species-specific HRP conjugated secondary antibodies (Dianova).

Techniques: Infection, Western Blot, Expressing, Control, Fractionation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: E3 ubiquitin ligase SKP2 limits autophagosome formation during Staphylococcus aureus infection

doi: 10.3389/fcimb.2026.1749151

Figure Lengend Snippet: Regulation and localization of SKP2 upon S. aureus infection. (A) Changes in SKP2 mRNA were analyzed by qPCR in A549 cells infected with S. aureus USA300 (MOI 10) at the depicted time points, fold change was calculated to 2 h uninfected control and normalized to the RPL47 housekeeping gene, n = 3. (B) SKP2 was immunoprecipitated from A549 cells infected with S. aureus USA300 (MOI 10) for 5 h and analyzed with anti-SKP2 and anti-acetyl lysine antibody compared to uninfected controls, heavy chain is indicated as unspecific band from the precipitating SKP2 antibody, n = 2. (C) THP-1 cells were treated with 25 µM of the p300-specific inhibitor C646 or left untreated and subsequently infected with S. aureus USA300 (MOI 10) for the depicted time points. SKP2 and PARP cleavage was analyzed by immunoblot compared to uninfected controls with β-actin as loading control, n = 2, one representative blot is shown. (D) SKP2 was immunoprecipitated from nuclear and cytoplasmic fractions of A549 cells 5 h post-infection with S. aureus USA300 (MOI 10) and analyzed by immunoblotting using anti-SKP2 and anti–acetyl-lysine antibodies and compared to uninfected cells. Lamin A and MEK1/2 were used as markers for the nuclear and cytoplasmic fractions, respectively, n = 2. (E) Analysis of nuclear and cytoplasmic fractionation for SKP2 abundance in A549 cells at 5 h post infection with S. aureus USA300 (MOI 10) with Lamin A serving as control for the nuclear fraction and MEK 1/2 serving as control for the cytoplasmic fraction, n = 3. (F) Immunofluorescence analysis of A549 cells infected for 5 h with S. aureus USA300 (MOI 10). Nuclei are stained with DAPI (blue), F-actin filaments with phalloidin (red), and SKP2 with an anti-SKP2-Alexa488 antibody (green), magnification 60x, scalebar 20 µm, n = 2.

Article Snippet: For immunoblotting, proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with primary antibodies against SKP2 (#2652, LOT:5), IκBα (#4814, LOT:21), LC3 (#12741, LOT:5), p27 (#3686, LOT:8), β-actin (#3700, LOT:21), lamin A (#86846, LOT:3), MEK1/2 (#4694, LOT:2; all from Cell Signaling Technology), anti-acetyl lysine antibody (#ab21623, LOT: 1010926-6; Abcam) and subsequently detected using species-specific HRP conjugated secondary antibodies (Dianova).

Techniques: Infection, Control, Immunoprecipitation, Western Blot, Fractionation, Immunofluorescence, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: E3 ubiquitin ligase SKP2 limits autophagosome formation during Staphylococcus aureus infection

doi: 10.3389/fcimb.2026.1749151

Figure Lengend Snippet: S. aureus protein A (SpA) contributes to increased SKP2 levels. (A) A549 epithelial cells treated with 50 µg/ml and 100 µg/ml SpA for the depicted timepoints were analyzed for SKP2 levels by immunoblotting with β-actin as loading control. (B) Densitometric analysis of SKP2 from immunoblot data, normalized to β-actin. Relative expression was calculated to the untreated control, which is indicated by the dotted line, n = 3. (C) A549 epithelial cells were infected with S. aureus USA300 (MOI 10) and USA300Δ spa (MOI 10) for the depicted time points and analyzed for SKP2 expression by immunoblotting with β-actin as loading control. (D) Densitometric analysis of SKP2 expression normalized to β-actin, relative expression was calculated to the 3 h uninfected control, n = 4. Data in B and D are presented as mean ± SD (*p < 0.05; **p < 0.01; ****p < 0.0001, students t-test).

Article Snippet: For immunoblotting, proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with primary antibodies against SKP2 (#2652, LOT:5), IκBα (#4814, LOT:21), LC3 (#12741, LOT:5), p27 (#3686, LOT:8), β-actin (#3700, LOT:21), lamin A (#86846, LOT:3), MEK1/2 (#4694, LOT:2; all from Cell Signaling Technology), anti-acetyl lysine antibody (#ab21623, LOT: 1010926-6; Abcam) and subsequently detected using species-specific HRP conjugated secondary antibodies (Dianova).

Techniques: Western Blot, Control, Expressing, Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: E3 ubiquitin ligase SKP2 limits autophagosome formation during Staphylococcus aureus infection

doi: 10.3389/fcimb.2026.1749151

Figure Lengend Snippet: Increased intracellular S. aureus and autophagy induction upon siRNA-mediated reduction of SKP2. A549 cells treated with SKP2-siRNA (siSKP2) or non-targeted control (NTC) siRNA were infected with S. aureus HG001 (MOI 20) for 5 h (A) Intracellular bacterial burden was determined as CFU normalized to 1 × 10 5 host cells, n = 4. (B) Cell lysates were analyzed for LC3-I, LC3-II, SKP2, and the SKP2 target p27 as a knockdown control, by immunoblotting with β-actin as loading control. (C–E) Densitometric analysis of p27, SKP2, and LC3-II normalized to β-actin, relative expression was calculated to NTC control, n = 4. Data in (A, C–E) are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001, students t-test).

Article Snippet: For immunoblotting, proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with primary antibodies against SKP2 (#2652, LOT:5), IκBα (#4814, LOT:21), LC3 (#12741, LOT:5), p27 (#3686, LOT:8), β-actin (#3700, LOT:21), lamin A (#86846, LOT:3), MEK1/2 (#4694, LOT:2; all from Cell Signaling Technology), anti-acetyl lysine antibody (#ab21623, LOT: 1010926-6; Abcam) and subsequently detected using species-specific HRP conjugated secondary antibodies (Dianova).

Techniques: Control, Infection, Knockdown, Western Blot, Expressing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: E3 ubiquitin ligase SKP2 limits autophagosome formation during Staphylococcus aureus infection

doi: 10.3389/fcimb.2026.1749151

Figure Lengend Snippet: Reduced intracellular S. aureus and formation of LC3-II in HeLa cells expressing SKP2 K145R K228R . HeLa cells transfected for 24 h with SKP2 K145R K228R (pcDNA SKP2mut) or empty vector control (pcDNA) were infected with S. aureus HG001 (MOI 20) for 3 h (A) Intracellular bacterial burden was determined as CFU normalized to 1 × 10 5 host cells, n = 3. (B) SKP2, LC3-I and LC3-II abundance was assessed via immunoblot with β-actin as loading control. (C, D) Densitometric analysis of LC3-II and SKP2 normalized to β-actin, relative expression was calculated to the uninfected control, n = 3. (E) HeLa cells transfected for 24 h with SKP2 K145R K228R (pcDNA SKP2mut) or empty vector control (pcDNA) were treated with 500 nM rapamycin alone or in combination with 100 nM bafilomycin A1 for 6 h or left untreated and subsequently analyzed for SKP2, LC3-I and LC3-II abundance via immunoblot with β-actin as loading control. (F) Densitometric analysis of LC3-II formation normalized to β-actin, relative expression was calculated to the untreated vector control, n = 3. Data are presented as mean ± SD. Data in (A, C, D, F) are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001, students t-test).

Article Snippet: For immunoblotting, proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with primary antibodies against SKP2 (#2652, LOT:5), IκBα (#4814, LOT:21), LC3 (#12741, LOT:5), p27 (#3686, LOT:8), β-actin (#3700, LOT:21), lamin A (#86846, LOT:3), MEK1/2 (#4694, LOT:2; all from Cell Signaling Technology), anti-acetyl lysine antibody (#ab21623, LOT: 1010926-6; Abcam) and subsequently detected using species-specific HRP conjugated secondary antibodies (Dianova).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Infection, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: E3 ubiquitin ligase SKP2 limits autophagosome formation during Staphylococcus aureus infection

doi: 10.3389/fcimb.2026.1749151

Figure Lengend Snippet: Stabilization of SKP2 attenuates autophagosome formation and prevents pathogen exploitation of the host autophagy machinery. Following pathogen internalization, pattern-recognition signals initiate autophagy induction and phagophore formation. Upon host cell entry, S. aureus is ubiquitinated on its surface, facilitating recognition by autophagy receptors. Upstream signaling through the Unc-51-like kinase 1 (ULK1) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (PI3KC3) complexes initiates phagophore formation, which then engulfs ubiquitinated bacteria. The autophagic machinery recruits the bacteria into forming autophagosomes. Three main outcomes are shown: (1) successful delivery of S. aureus to lysosomes leads to bacterial elimination, or (2) S. aureus escapes from the autophagosome, allowing replication within the host cytosol, or (3) block in lysosomal fusion and replication within the phagosome. Under conditions in which SKP2 is stabilized, acetyltransferase p300-mediated acetylation increases SKP2 levels and prevents its degradation. Elevated SKP2 limits autophagosome formation preventing exploitation of the phagosome by S. aureus .

Article Snippet: For immunoblotting, proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with primary antibodies against SKP2 (#2652, LOT:5), IκBα (#4814, LOT:21), LC3 (#12741, LOT:5), p27 (#3686, LOT:8), β-actin (#3700, LOT:21), lamin A (#86846, LOT:3), MEK1/2 (#4694, LOT:2; all from Cell Signaling Technology), anti-acetyl lysine antibody (#ab21623, LOT: 1010926-6; Abcam) and subsequently detected using species-specific HRP conjugated secondary antibodies (Dianova).

Techniques: Bacteria, Blocking Assay