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

Journal: Cell Death & Disease

Article Title: Targeting FOXM1 regulates metabolic signatures through ROS-dependent JNK/Bmi1/Skp2 axis in human cutaneous T-cell lymphoma

doi: 10.1038/s41419-025-08389-z

Figure Lengend Snippet: HH and H9 cells were treated with TST(5 μM) and SP600125 (10 μM), either alone or in combination. Following treatment, cell lysates were prepared, and western blotting was performed. A – L show the immunoblotting results for FOXM1, KLF4, Bmi1, Skp2, and p21, along with their densitometric quantification data presented as mean ± SD ( n = 3). Band intensities were normalized to the respective loading control and quantified using Image Lab software. Statistical significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 compared with the respective control (positive and negative) groups. M , N Demonstrate that JNK inhibition reversed TST-mediated suppression of spheroid formation in HH and H9 cells.

Article Snippet: Different antibodies, including Phospho-JNK (Thr183/Tyr185) (Cat# 4668), JNK (Cat# 9252), FOXM1 (Cat# 20459), Caspase-3 (Cat# 9662), Cleaved caspase-3 (Cat# 9661), Cleaved caspase-8 (Cat# 9496), Skp2 (Cat# 4358), Bmi1(Cat# 6964), p21 (Cat# 64016), PARP (Cat# 9542), Cleaved PARP (Cat# 9544), LC3A/B (Cat# 4108), GAPDH (Cat# 5174), and β-actin (Cat# 4967), were procured from Cell Signaling Technology, Inc. (3 Trask Lane Danvers, MA 01923).

Techniques: Western Blot, Control, Software, Inhibition