Journal: International Dental Journal

Article Title: Hypoxia-Inducible Factor-1α Stabilization Alleviates Hypoxia-Induced Temporomandibular Joint Osteoarthritis by Activating MDM2 to Suppression of p53 Signalling

doi: 10.1016/j.identj.2026.109805

Figure Lengend Snippet: DMOG-mediated HIF-1α stabilization exerts a protective effect by inhibiting p53 signalling transduction. (A) Volcano plot of DEGs in chondrocytes treated with or without DMOG ( P < .05, |fold change| > 2) under hypoxic conditions. (B) KEGG pathway enrichment analysis of DEGs. (C) Representative immunohistochemical images of p53 in condylar chondrocytes (scale bar: 100 μm) and quantitative analysis of p53-positive area ( n = 6). (D and E) Western blot and quantitative analysis of HIF-1α, p53, p21, and BAX protein levels in chondrocytes under hypoxia and hypoxia + DMOG conditions. (F and G) Western blot images and quantitative analysis of iNOS, HIF-1α, MMP13, p53, p21, BAX, and TNF-α protein levels in chondrocytes under hypoxia, hypoxia + DMOG, and hypoxia + DMOG + NSC-207895 (HX + DMOG + NSC-207895) conditions ( n = 3). (H) Immunofluorescence staining images and quantitative analysis of IL-6 and MMP3 in chondrocytes treated with DMOG and NSC-207895 under hypoxia ( n = 3). Scale bar: 50 μm. Data are presented as mean ± SEM. Statistical significance was determined by unpaired Student’s t test (for pairwise comparisons) or one-way ANOVA (for multigroup comparisons), as appropriate. ns, no significance, * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: The membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies: HIF-1α (1:1000; Abcam), p53 (1:1000; ABclonal), TNF-α (1:1000; Huabio), MMP13 (1:1000; Abcam), iNOS (1:1000; Servicebio), BAX (1:1000; Proteintech), p21 (1:1000; Abcam), MDM2 (1:1000; Immunoway), and α-Tubulin (1:5000; Servicebio).

Techniques: Transduction, Immunohistochemical staining, Western Blot, Immunofluorescence, Staining

Journal: International Dental Journal

Article Title: Hypoxia-Inducible Factor-1α Stabilization Alleviates Hypoxia-Induced Temporomandibular Joint Osteoarthritis by Activating MDM2 to Suppression of p53 Signalling

doi: 10.1016/j.identj.2026.109805

Figure Lengend Snippet: Stabilized HIF-1α transcriptionally activates Mdm2 to promote p53 ubiquitin-proteasomal degradation in hypoxic condylar. (A) Co-IP assay detecting endogenous p53 ubiquitination in hypoxic chondrocytes treated with or without 1 mM DMOG. (B and C) qRT-PCR analysis of Tp53 and Mdm2 mRNA expression in chondrocytes cultured under hypoxia with or without DMOG intervention. (D) Reciprocal Co-IP assay for detecting physical interaction between MDM2 and p53 in hypoxic chondrocytes with or without DMOG treatment. (E) Western blot analysis of p53 protein abundance in hypoxic chondrocytes incubated with DMOG in the presence or absence of 10 μM MG132. (F) JASPAR database-based in silico prediction of potential HIF-1α binding sites on rat Mdm2 promoter. (G) ChIP-qPCR assay measuring HIF-1α occupancy on the Mdm2 promoter in hypoxic chondrocytes with or without DMOG. (H) Schematic diagram. Data are presented as mean ± SEM ( n = 3 per group). Statistical significance was determined by unpaired Student’s t test. ** P < .01, *** P < .001.

Article Snippet: The membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies: HIF-1α (1:1000; Abcam), p53 (1:1000; ABclonal), TNF-α (1:1000; Huabio), MMP13 (1:1000; Abcam), iNOS (1:1000; Servicebio), BAX (1:1000; Proteintech), p21 (1:1000; Abcam), MDM2 (1:1000; Immunoway), and α-Tubulin (1:5000; Servicebio).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Quantitative Proteomics, Incubation, In Silico, Binding Assay, ChIP-qPCR

Journal: Translational Cancer Research

Article Title: ALOX12 suppresses colon cancer progression by promoting p53-mediated ferroptosis through upregulating ROS-induced stress

doi: 10.21037/tcr-2026-0503

Figure Lengend Snippet: ALOX12 is essential for p53-mediated ferroptosis in CC. (A) Western blotting and qRT-PCR analysis of HCT116 and DLD-1 cells incubated with Nutlin-3 (0, 10, 15, 20, 30, 40 µM) for 24 h. Mean ± SD, n=3 independent experiments. (B) Representative phase-contrast images of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Scale bars, 100 µm. N=3 independent experiments. (C) Cell death analysis of HCT116 and DLD-1 cells. HCT116 cells incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h. Mean ± SD, n=3 independent experiments. (D-F) ROS (D), MDA (E) and Fe 2+ (F) detection of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. ** P<0.01; ****, P<0.0001; ns, not significant. CC, colon cancer; MDA, malondialdehyde; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROS, reactive oxygen species; SD, standard deviation; TBH, tert-Butyl hydroperoxide.

Article Snippet: Protein extracts were analyzed by western blotting according to standard protocols, using primary antibodies against p53 (A0263, Abclonal, Wuhan, China, 1:1,000), SLC7A11 (CY7046, Abways, Shanghai, China, 1:1,000), ALOX12 (CY8070, Abways, 1:1,000), GAPDH (AB0037, Abways, 1:5,000), and GPX4 (A11243, Abclonal, 1:1,000), followed by an HRP-conjugated goat anti-rabbit secondary antibody (AS014, Abclonal, 1:5,000).

Techniques: Western Blot, Quantitative RT-PCR, Incubation, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Journal: Translational Cancer Research

Article Title: ALOX12 suppresses colon cancer progression by promoting p53-mediated ferroptosis through upregulating ROS-induced stress

doi: 10.21037/tcr-2026-0503

Figure Lengend Snippet: Mechanisms into the regulation of ALOX12 in p53-mediated ferroptosis. (A) Western blotting analysis of HCT116, DLD-1 cells transfected with ALOX12 lentivirus or si-ALOX12 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (B) Western blotting analysis of HCT116 and DLD-1 cells transfected with si-p53 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (C) Western blotting analysis of HCT116 and DLD-1 cells treated with Nutlin-3 (10 µM) or co-transfected with si-p53 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (D) Cell death analysis of HCT116 and DLD-1 cells. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (E-G) ROS (E), MDA (F) and Fe 2+ (G) detection of HCT116 and DLD-1 cells. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (H) Cell death analysis of HCT116 and DLD-1 cells when considering ALOX12 status. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (I) Co-IP assay of HCT116 and DLD-1 cells using SLC7A11 antibody (see methods). N=3 independent experiments. (J) Co-IP assay of HCT116 and DLD-1 cells using anti-Flag antibody (see Methods). N=3 independent experiments. (K) ALOX12 enzyme activity analysis of HCT116 and DLD-1 cells by ELISA (see Methods), cells were pre-treated with ML355 (2 µM), Nutlin-3 (10 µM) or co-transfected with si-p53 for 24 h. Mean ± SD, n=3 independent experiments. (L,M) MDA (L) and Fe 2+ (M) detection of HCT and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), Lip-1 (2 µM) or transfected with si-p53 for 24 h, while DLD-1 cells were treated with Nutlin-3 (10 µM), TBH (100 µM), Lip-1 (2 µM) or transfected with si-p53 for 24 h. Mean ± SD, n=3 independent experiments. *, P<0.05; ** P<0.01; *** P<0.001; ****, P<0.0001; ns, not significant. Co-IP, co-immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; MDA, malondialdehyde; ROS, reactive oxygen species; SD, standard deviation; TBH, tert-Butyl hydroperoxide.

Article Snippet: Protein extracts were analyzed by western blotting according to standard protocols, using primary antibodies against p53 (A0263, Abclonal, Wuhan, China, 1:1,000), SLC7A11 (CY7046, Abways, Shanghai, China, 1:1,000), ALOX12 (CY8070, Abways, 1:1,000), GAPDH (AB0037, Abways, 1:5,000), and GPX4 (A11243, Abclonal, 1:1,000), followed by an HRP-conjugated goat anti-rabbit secondary antibody (AS014, Abclonal, 1:5,000).

Techniques: Western Blot, Transfection, Expressing, Incubation, Co-Immunoprecipitation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Standard Deviation

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: Iron accumulation impairs mitophagy, promotes senescence, and suppresses osteogenic differentiation in BMSCs. (a) Schematic diagram of extraction of BMSCs from human femur. (b) Western blot analysis of osteogenic marker proteins (RUNX2, ALP) in BMSCs from normal controls and postmenopausal osteoporosis patients and osteoporosis patients with iron accumulation. (c) Alizarin Red S (ARS) staining of BMSCs treated with increasing concentrations of FAC (0, 50, 100, 200 μM) for 21 days and alkaline phosphatase (ALP) staining of BMSCs treated with increasing concentrations of FAC (0, 50, 100, 200 μM) for 14 days. Scale bar: 50 μm. (d) Western blot analysis of osteogenic markers (RUNX2, ALP) in FAC-treated BMSCs for 5 days. (e) RT-qPCR analysis of osteogenic genes ( Runx2, Alpl, Bglap, Sp7 ) in FAC-treated BMSCs for 72h. (f) KEGG pathway enrichment analysis of differentially expressed genes from RNA sequencing of control and 200 μM FAC-treated BMSCs for 72h. (g, h) Immunofluorescence staining of senescence markers (γ-H2AX, H3K9me3) in FAC-treated BMSCs for 72h. Scale bar: 20 μm. (i) Senescence-associated β-galactosidase (SA-β-gal) staining of FAC-treated BMSCs for 72h. Scale bar: 50 μm. (j) Flow cytometric quantification of SA-β-gal activity in FAC-treated BMSCs for 72h. (k) Western blot analysis of senescence-related proteins (P53, P21, P16) in FAC-treated BMSCs for 72h. (l) Mitophagy assessment by immunofluorescence co-staining with Mitophagy Dye (red) and MitoTracker (green) in FAC-treated BMSCs for 72h. Scale bar: 20 μm. (m) Western blot analysis of mitophagy/autophagy-related proteins (PINK1, PARKIN, P62, LC3) in FAC-treated BMSCs for 72h. (n) Mitochondrial membrane potential (MMP) detection by MT-1 staining in FAC-treated BMSCs for 72h. Scale bar: 30 μm. Data are presented as mean ± SEM; One-way ANOVA (Dunnett's multiple-comparison test); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

Techniques: Extraction, Western Blot, Marker, Staining, Quantitative RT-PCR, RNA Sequencing, Control, Immunofluorescence, Activity Assay, Membrane, Comparison

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: Mitophagy activation rescues iron accumulation-induced mitochondrial dysfunction, cellular senescence, and impaired osteogenic differentiation in BMSCs. BMSCs were isolated from normal mice and treated with 200 μM FAC with or without CCCP co-treatment for the same duration in each assay. The time points for the indicated assays were the same as those in . (a) Western blot analysis of mitophagy/autophagy-related proteins (PINK1, PARKIN, P62, LC3). (b, c) Flow cytometric analysis of (b) intracellular ROS and (c) mitochondrial superoxide levels. (d) Mitochondrial membrane potential assessment by MT-1 immunofluorescence staining. Scale bar: 30 μm. (e) Cellular ATP content measurement. (f – i) Immunofluorescence analysis of senescence markers (f, h) γ-H2AX and (g, i) H3K9me3. Scale bar: 40 μm. (j) Western blot analysis of senescence-related proteins (P53, P21, P16). (k) Alizarin Red S (ARS) and alkaline phosphatase (ALP) staining. Scale bar: 50 μm. (l) Western blot analysis of osteogenic marker proteins (RUNX2, ALP). Data are presented as mean ± SEM; One-way ANOVA (Tukey's multiple-comparison test); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

Techniques: Activation Assay, Isolation, Western Blot, Membrane, Immunofluorescence, Staining, Marker, Comparison

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: Mitophagy activation alleviates BMSC senescence and restores bone mass in iron-accumulating mice. (a) Representative micro-CT images of distal femoral trabecular bone. (b) Quantitative micro-CT analysis of trabecular bone parameters: Tb.BMD (trabecular bone mineral density), BV/TV (bone volume fraction), BS/TV (bone surface density), and Tb.N (trabecular number). (c) Detection of the serum OCN and P1NP levels from the mice in each group. (d) Histological analysis of tibial sections via H&E staining, toluidine blue staining, and DAPI immunofluorescence from the mice in each group. Scale bar: 250 μm. (e) Detection of the bone formation rate by calcein double labeling from the mice in each group. Scale bar: 20 μm. (f – i) Immunofluorescence analysis of senescence markers (γ-H2AX and H3K9me3) in BMSCs isolated from different treatment groups. Scale bar: 50 μm. (j) Western blot analysis of senescence-related proteins (P53, P21, P16) in BMSCs. (k) Western blot analysis of mitophagy/autophagy-related proteins (PINK1, PARKIN, P62, LC3) in BMSCs. (l) Mitochondrial membrane potential assessment by MT-1 immunofluorescence staining in BMSCs. Scale bar: 50 μm. (m) Cellular ATP content measurement in BMSCs. (n – o) Flow cytometric analysis of (n) intracellular ROS and (o) mitochondrial superoxide levels in BMSCs. Data are presented as mean ± SEM; One-way ANOVA (Tukey's multiple-comparison test); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

Techniques: Activation Assay, Micro-CT, Staining, Immunofluorescence, Labeling, Isolation, Western Blot, Membrane, Comparison

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: PINK1 overexpression rescues iron accumulation-induced mitochondrial dysfunction, senescence, and osteogenic impairment in BMSCs. The time points for the indicated assays were the same as those in . (a) Western blot analysis of mitophagy/autophagy-related proteins (PINK1, PARKIN, P62, LC3) in BMSCs transduced with control or PINK1-overexpressing lentivirus followed by FAC treatment. (b) Mitochondrial membrane potential assessment by MT-1 immunofluorescence staining. Scale bar: 50 μm. (c) Cellular ATP content measurement. (d, e) Flow cytometric analysis of (d) intracellular ROS and (e) mitochondrial superoxide levels. (f) Western blot analysis of senescence-related proteins (P53, P21, P16). (g – j) Immunofluorescence analysis of senescence markers (γ-H2AX and H3K9me3). Scale bar: 50 μm. (k, l) Alizarin Red S (ARS) staining and Alkaline phosphatase (ALP) staining. Scale bar: 50 μm. (m) Western blot analysis of osteogenic marker proteins (RUNX2, ALP). (n) RT-qPCR analysis of osteogenic genes ( Runx2, Alpl, Bglap, Sp7 ). Data are presented as mean ± SEM; One-way ANOVA (Tukey's multiple-comparison test); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

Techniques: Over Expression, Western Blot, Transduction, Control, Membrane, Immunofluorescence, Staining, Marker, Quantitative RT-PCR, Comparison

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: Impaired mitophagy in BMSCs from osteoporosis patients with iron accumulation. (a) Western blot analysis of senescence-related proteins (P53, P21, P16) in BMSCs from normal controls, postmenopausal osteoporosis patients and osteoporosis patients with iron accumulation. (b) Western blot analysis of mitochondrial ferritin (FTMT) expression levels in BMSCs. (c) Western blot analysis of mitophagy/autophagy-related proteins PINK1, p-PINK1(Ser228), PARKIN, P62, and LC3 in BMSCs. (d) Western blot analysis of mitophagy/autophagy-related proteins PINK1, PARKIN, P62, and LC3 in BMSCs of PMOP and IOP group with or without CCCP intervention. (e) Western blot analysis of senescence-related proteins (P53, P21, P16) in BMSCs of PMOP and IOP group with or without CCCP intervention. (f) Western blot analysis of osteogenic marker proteins (RUNX2, ALP) in BMSCs of PMOP and IOP group with or without CCCP intervention.

Article Snippet: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

Techniques: Western Blot, Expressing, Marker

Journal: Tumour Virus Research

Article Title: HPV18E6 and CDK5 virus-host interaction is a prospective therapeutic target for HPV-positive cervical cancer

doi: 10.1016/j.tvr.2026.200339

Figure Lengend Snippet: A mutualistic association of CDK5 and 18E6 proteins. (A) (i). The represented immunoblot image of CDK5 upregulates the level of 18E6 protein. HEK 293 cells were transfected with an increasing amount of pcDNA3.1: His-CDK5 (1, 2, and 4 μg) and a fixed amount of pcDNA3.1: HA-18E6 (2 μg). (ii). The represented immunoblot image of 18E6 upregulates the level of CDK5 protein. HEK 293 cells were transfected with an increasing amount of pcDNA3.1: HA-18E6 (1, 2, and 4 μg) and pcDNA3.1: His-CDK5 (2 μg). (B) (i). The represented immunoblot image of CDK5 upregulates the level of 18E6 protein in the HeLa cell lines. HeLa cells were transfected with an increasing amount of pcDNA3.1: His-CDK5 (1, 2, 3, and 4 μg). (ii). The represented immunoblot image of 18E6 upregulates the level of CDK5 protein in the SAS cell lines. SAS cells were transfected with an increasing amount of pcDNA3.1: HA-18E6 (1, 2, 3, and 4 μg). (C) (i). The represented immunoblot image of CDK5, E6AP, and p53 after silencing 18E6/E7 expression. Total protein lysates were extracted from HeLa cell lines transfected with siRNA against control (siCtrl) or 18 E6 and E7 (si18 E6/E7) for 72 h. (ii-v) The relative expression levels of 18E6, CDK5, p53, and E6AP relative to β-actin and analyzed using ImageJ and GraphPad Prism (n = 3). All data were presented as means ± standard error of the mean (SEM). (∗, P < 0.05; ∗∗, P < 0.01). (D) The represented immunoblot of CDK5, pCDK5 and HPV 18E6 proteins in the stable expression of HPV 18 in the HGK12 cell line (HPV-null primary keratinocytes cell line), HC: Treatment with 0.2 μM CP681301; LC: Treatment with 0.1 μM CP681301.

Article Snippet: The transferred membranes were incubated overnight at 4 °C with the following primary antibodies: monoclonal rabbit anti-HA (Cell Signaling Technology), monoclonal mouse anti-p53 (Santa Cruz), monoclonal mouse anti-HPV18E6 (Santa Cruz), monoclonal mouse anti-CDK5 (Santa Cruz), and monoclonal mouse anti-β-actin (Santa Cruz).

Techniques: Western Blot, Transfection, Expressing, Control

Journal: Tumour Virus Research

Article Title: HPV18E6 and CDK5 virus-host interaction is a prospective therapeutic target for HPV-positive cervical cancer

doi: 10.1016/j.tvr.2026.200339

Figure Lengend Snippet: CP681301 treatment destabilized E6, reduced E6AP and rescued p53. (A). HeLa and C33A cell lines were treated with CP681301 at the concentrations of 0.6 μM and 1.5 μM for 24 h. The protein extracts were subjected to Western blotting for the detection of CDK5, 18E6, E6AP, p53, and β-actin proteins. (B). CP681301 disrupted the 18E6-E6AP protein complex. The inhibition of CP681301 was detected using the GST-pull down assay, where CP681301 was incubated with the purified GST-18E6 protein and Flag-E6AP protein for 2h. After extensive washing, the bound E6AP protein was detected via Western blotting using an anti-Flag antibody. The immunoblot (IB) on the upper panel shows the interaction of E6AP with GST-18E6, while the lower panel shows the Ponceau S stained of the blot.

Article Snippet: The transferred membranes were incubated overnight at 4 °C with the following primary antibodies: monoclonal rabbit anti-HA (Cell Signaling Technology), monoclonal mouse anti-p53 (Santa Cruz), monoclonal mouse anti-HPV18E6 (Santa Cruz), monoclonal mouse anti-CDK5 (Santa Cruz), and monoclonal mouse anti-β-actin (Santa Cruz).

Techniques: Western Blot, Inhibition, Pull Down Assay, Incubation, Purification, Staining