Journal: International Journal of Molecular Medicine

Article Title: Novel Hsp90 inhibitor JD-02 inhibits HSV-1 infection via the Raf/MEK/ERK signaling pathway

doi: 10.3892/ijmm.2026.5810

Figure Lengend Snippet: JD-02 inhibits HSV-1 replication by suppressing the Raf/MEK/ERK signaling pathway. (A) Western blot analysis was conducted to assess the effects of HSV-1 (MOI=0.1) infection on the protein levels of BRAF, MEK and ERK, with and without treatment using JD-02. (B) Treatment with JD-02 at the specified concentration influences the protein levels of BRAF, MEK and ERK in HaCaT cells over a 12-h period. (C and D) HaCaT cells were subjected to transfection with either N.C. siRNA or ERK siRNA for a period of 48 h. Subsequently, the cells were infected with HSV-1 (MOI=0.1) for an additional 24 h. The DNA copy number of the viral gene UL54, as well as the viral protein expression of gB, ICP0, ICP27, ERK and p-ERK were evaluated. (E) The DNA copy numbers of the viral genes UL30 , UL52 and UL54 in HaCaT cells infected with HSV-1 (MOI=0.1) and subsequently treated with either JD-02 (1 μ M) or U0126 (10 μ M) for 24 h, were quantified using reverse transcription-quantitative PCR. (F) Western blot analysis of viral proteins (gB, ICP0 and ICP27), ERK and p-ERK expression in HaCaT cell infected with HSV-1 (MOI=0.1) and treated with U0126 for indicated concentration. (G) Western blot analysis of UL30 overexpression in HaCaT cells treated with U0126. Data are presented as the mean ± SD (n=3). * P<0.05, ** P<0.01, *** P<0.01 and **** P<0.0001 compared with the HSV-1 group. HSV, Herpes Simplex Virus; MOI, multiplicity of infection; N.C., negative control; siRNA, small interfering RNA; p-, phosphorylated.

Article Snippet: These primary antibodies included ICP0 (1:1,000; cat. no. ab6513; Abcam), ICP27 (1:1,000; cat. no. ab53480; Abcam), gD (1:1,000; cat. no. ab6507; Abcam), gB (1:500; cat. no. sc-56987; Santa Cruz Biotechnology, Inc.), Raf-B (1:500; cat. no. sc-166; Santa Cruz Biotechnology, Inc.), p-BRAF (1:1,000; cat. no. 2696T; Cell Signaling Technology, Inc.), ERK (1:1,000; cat. no. 4695S; Cell Signaling Technology, Inc.), p-ERK (1:1,000; cat. no. 4370S; Cell Signaling Technology, Inc.), MEK1/2 (1:1,000; cat. no. AF6385; Affinity Biosciences), p-MEK1/2 (1:1,000; cat. no. 9154T; Cell Signaling Technology, Inc.), Flag (1:1,000; cat. no. 14793S; Cell Signaling Technology, Inc.), HA (1:1,000; cat. no. 3724S; Cell Signaling Technology, Inc.).

Techniques: Western Blot, Infection, Concentration Assay, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Over Expression, Virus, Negative Control, Small Interfering RNA

Journal: Oncology Letters

Article Title: Methylglyoxal-derived glycated albumin enhances the stemness potential of invasive ductal carcinoma-derived breast cancer stem-like cell line KAIMRC1

doi: 10.3892/ol.2026.15541

Figure Lengend Snippet: GA effects on ERK1/2 phosphorylation in invasive ductal carcinoma-derived KAIMRC1 stem-like cells. Representative western blot analyses showed p-ERK1/2 in KAIMRC1 stem-like cells treated with 100 µg/ml GA for (A) 30 to 120 min and with (B) 50–100 µg/ml GA or unglycated BSA for 90 min of incubation. Bar graphs show the relative expression level of p-ERK1/2 normalized to total ERK1/2. GAPDH served as the loading control. Data are presented as the mean ± SD from three independent experiments. Statistical significance relative to the untreated control is indicated as *P < 0.05 and **P < 0.01. C, control; GA, glycated albumin; p-ERK1/2, phosphorylated-ERK1/2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Mouse primary monoclonal antibodies directed against extracellular signal-regulated kinase (ERK)1 (clone G-8; cat. no. sc-271269), phosphorylated-ERK1/2 (p-ERK1/2; clone E-4; Tyr204 of ERK1; cat. no. sc-7383), OCT3/4 (clone A-9; cat. no. sc-365509) and RAGE (clone E-1; cat. no. sc-74473) were obtained from Santa Cruz Biotechnology, Inc.

Techniques: Phospho-proteomics, Derivative Assay, Western Blot, Incubation, Expressing, Control

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: SERPINE1-mediated modulation of the ERK/p38 ratio and anoikis. (A) Upper panel: western blotting showing ERK, p-ERK, p38, and p-p38 levels. Quantitative data showed the p-ERK/ERK and p-p38/p38 ratios in SERPINE1 knockdown cells compared with control cells. Lower panel: western blotting analysis of p-AKT, AKT, p-JNK and JNK (normalized to total protein) levels in the shSE1 and shc groups. The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins. Data represent mean ± SDs of three independent experiments. (B) PLAUR and HSP90AA1 mRNA and protein levels in shSE1 and shc cells analyzed using RNA-seq (left panel) and western blotting (right panel). The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins and represent mean ± SDs of three independent experiments. RNA-seq was performed using one biological replicate per group. (C-D) Upper panel: (C) 231-shSE1 cells were infected with lentivirus containing PLAUR cDNA (ex-PLAUR) or the corresponding empty vector control (vec). (D) H4-shSE1 cells were transfected with siRNA targeting HSP90AA1 (si-HSP90AA1) or siNC. The expression levels of uPAR and HSP90α, as well as the activity of ERK and p38, were detected by western blotting, with GAPDH serving as the loading control. Data represent mean ± SDs of three independent experiments. Lower panel: Cell proliferation was assessed by CCK-8 assay in 231-shSE1 cells infected with ex-PLAUR or vec, and in H4-shSE1 cells transfected with si-HSP90AA1 or siNC. Statistical significance was determined using two-way ANOVA followed by Šídák's multiple comparisons test. The data are presented as the means ± SDs. n=6. (E) Morphology of shSE1 and shc cells cultured under suspension condition. (scale bar, 100 μ m). (F) Apoptotic cells cultured under suspension condition were analyzed by flow cytometry following Annexin V-PE/7AAD staining (72 h of suspension culture). Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs of three independent experiments. ** P<0.01 *** P<0.001, ns, not significant. SERPINE1, serine protease inhibitor clade e member 1; p-, phosphorylated; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; shc, shRNA scrambled control; PLAUR, plasminogen activator, urokinase receptor; HSP90AA1, heat shock protein 90 alpha family class a member 1; RNA-seq, RNA sequencing; vec, empty vector control; siRNA, short interfering RNA; siNC, negative control siRNA; uPAR, urokinase-type plasminogen activator receptor; HSP90α, heat shock protein 90-alpha.

Article Snippet: Following the blocking of nonspecific binding sites using 5% skimmed milk (cat. no. P0216; Beyotime Biotechnology) or bovine serum albumin (cat. no. NGP0028A; Beyotime Biotechnology) for 1 h at room temperature, the membranes were incubated with primary antibodies (incubation overnight at 4°C) against SERPINE1 (cat. no. 13801-1-AP; Proteintech Group, Inc), GAPDH (cat. no. sc-25778; Santa Cruz Biotechnology, Inc.), MCM3 (cat. no. PA5-79646; Thermo Fisher Scientific, Inc.), phosphorylated (p-)MCM3 (Ser112; cat. no. TA2362; Abmart Pharmaceutical Technology Co., Ltd.), ERK (cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), uPAR (cat. no. ab10379; Abcam), Histone H3 (cat. no. 4499), p-p53 (Ser15; cat. no. 9284), p53 (cat. no. 2524), p-SMAD3 (Ser423/425; cat. no. 9520), SMAD3 (cat. no. 9523), p-Rb (Ser807/811; cat. no. 8516), Rb (cat. no. 9309), CyclinD1 (cat. no. 55506), CyclinE1 (cat. no. 20808), p21 (cat. no. 2947), p-p38 (cat. no. 4511), p38 (cat. no. 8690), p-ERK (cat. no. 4370), p-JNK (cat. no. 9255), JNK (cat. no. 9252), p-AKT (Ser473; cat. no. 4060), AKT (cat. no. 9272), HSP90α (cat. no. 4877) and MMP-1 (cat. no. 54376) were from Cell Signaling Technology, Inc.) and subsequently incubated with the corresponding secondary antibodies (dilution 1:4,000) for 2 h at room temperature.

Techniques: Western Blot, Knockdown, Control, Expressing, RNA Sequencing, Infection, Plasmid Preparation, Transfection, Activity Assay, CCK-8 Assay, Cell Culture, Suspension, Flow Cytometry, Staining, Protease Inhibitor, shRNA, Small Interfering RNA, Negative Control

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: SERPINE1 regulates MMP activity. (A) MMP and TIMP levels in the supernatants of shSE1 and shc cells after 24 h of incubation and 10-fold concentration. Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs. n=4. (B) Quantification of active MMP-1 and MMP-13 levels in cell lysates using fluorescence ELISA. Statistical significance was determined using two-way ANOVA followed by Šídák's multiple comparisons test. The data are presented as the means ± SDs of three independent experiments. (C) Western blotting showing the levels of the indicated proteins in H4-shSE1 cells at 72 h after transfection with the si-HSP90AA1 or siNC. The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins and represent mean ± SDs of three independent experiments. (D) Representative images and quantification of Transwell invasion assays through Matrigel and collagen type I in H4-shSE1 cells transfected with the si-HSP90AA1 or siNC (scale bar, 100 μ m). Statistical significance was determined using a two-sided Student's t-test. The data are presented as the means ± SDs of three independent experiments. (E) Diagram showing the mechanisms underlying the effects of SERPINE1 on cancer proliferation and invasion. SERPINE1 knockdown influences cell proliferation and invasion through distinct signaling pathways. With respect to proliferation, SERPINE1 knockdown reduces TGF-β levels, and this reduction alters the activity of SMAD3, p53, and MCM3 to promote cell cycle progression. SERPINE1 knockdown interferes with the uPAR-mediated balance of the ERK/p38 ratio; it may also affect this ratio by modulating HSP90α expression and p38 activity, which suppress cell proliferation. In terms of invasion, SERPINE1 downregulation increases MMP-1 levels via the HSP90α-p38 pathway, thereby promoting cellular invasion. *** P<0.001, ** P<0.01, ns, not significant. SERPINE1, serine protease inhibitor clade e member 1; MMP, matrix metalloproteinases; TIMP, tissue inhibitors of metal proteases; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; si, short interfering RNA; siHSP90AA1, short interfering heat shock protein 90 alpha family class a member 1; NC, negative control; uPAR, urokinase-type plasminogen activator receptor; p-, phosphorylated; MCM3, minichromosome maintenance complex component 3; HSP90α, heat shock protein 90-alpha.

Article Snippet: Following the blocking of nonspecific binding sites using 5% skimmed milk (cat. no. P0216; Beyotime Biotechnology) or bovine serum albumin (cat. no. NGP0028A; Beyotime Biotechnology) for 1 h at room temperature, the membranes were incubated with primary antibodies (incubation overnight at 4°C) against SERPINE1 (cat. no. 13801-1-AP; Proteintech Group, Inc), GAPDH (cat. no. sc-25778; Santa Cruz Biotechnology, Inc.), MCM3 (cat. no. PA5-79646; Thermo Fisher Scientific, Inc.), phosphorylated (p-)MCM3 (Ser112; cat. no. TA2362; Abmart Pharmaceutical Technology Co., Ltd.), ERK (cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), uPAR (cat. no. ab10379; Abcam), Histone H3 (cat. no. 4499), p-p53 (Ser15; cat. no. 9284), p53 (cat. no. 2524), p-SMAD3 (Ser423/425; cat. no. 9520), SMAD3 (cat. no. 9523), p-Rb (Ser807/811; cat. no. 8516), Rb (cat. no. 9309), CyclinD1 (cat. no. 55506), CyclinE1 (cat. no. 20808), p21 (cat. no. 2947), p-p38 (cat. no. 4511), p38 (cat. no. 8690), p-ERK (cat. no. 4370), p-JNK (cat. no. 9255), JNK (cat. no. 9252), p-AKT (Ser473; cat. no. 4060), AKT (cat. no. 9272), HSP90α (cat. no. 4877) and MMP-1 (cat. no. 54376) were from Cell Signaling Technology, Inc.) and subsequently incubated with the corresponding secondary antibodies (dilution 1:4,000) for 2 h at room temperature.

Techniques: Activity Assay, Incubation, Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Expressing, Knockdown, Protein-Protein interactions, Protease Inhibitor, shRNA, Small Interfering RNA, Negative Control