Journal: Plants (Basel, Switzerland)

Article Title: Enhancing Maize Transformation and Targeted Mutagenesis through the Assistance of Non-Integrating Wus2 Vector.

doi: 10.3390/plants12152799

Figure Lengend Snippet: Figure 1. NIW-assisted transformation. (a) Schematic illustration of NIW-assisted maize transforma- tion. Agrobacterium cells harboring GOI and NIW constructs are mixed at a 9:1 ratio before immature embryo infection, and Wus2 protein diffuses to the neighboring cells through the plasmodesmata and stimulates cell proliferation, promoting the regeneration of cells without Wus2 integration. (b) T-DNA region of NIW construct pKL2391. (c) T-DNA region of GOI construct pKL2359. LB and RB, left and right T-DNA border sequences; P35S-RUBY, RUBY reporter consisting of betalain biosynthesis genes (CYP76AD1, DODA, and Glucosyl transferase) driven by CaMV 35S promoter and Arabidopsis heat shock protein 18.2 gene terminator; 3×ENH-PzmUbi-ZmWUS2, maize Wus2 expression cassette driven by 3× viral enhancers, maize ubiquitin promoter, and In2-1 gene terminator; P35S-mCherry, a red fluorescent protein (mCherry) expression cassette driven by CaMV 35S promoter and soybean vegetative storage protein terminator; PzmUbi-SpCas9, maize codon optimized Cas9 from Streptococ- cus pyogenes (SpCas9) with maize ubiquitin promoter and rbcS-E9 gene terminator; PosU3-gRNA, a single-guide RNA targeting maize Glossy2 gene driven by rice U3 promoter; PzmUbi-NptII, neomycin phosphotransferase II (NptII) gene driven by maize ubiquitin promoter and potato proteinase inhibitor II gene terminator.

Article Snippet: LBA4404Thystrain was used for the GOI construct pKL2359 (Figure 1c, Addgene#199721; [12]) in all experiments.

Techniques: Transformation Assay, Construct, Infection, Expressing, Ubiquitin Proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: Overexpression of TRIM38 inhibits TNFα- and IL-1β–triggered signaling. (A) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation in HEK293 cells. HEK293 cells (1 × 105) were transfected with the NF-κB luciferase plasmid (0.01 μg) and an HA-TRIM38 plasmid (0.2 or 0.4 μg). Twenty hours after transfection, cells were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) or left untreated for 10 h before luciferase assays were performed. Expression of transfected TRIM38 in each unstimulated sample was examined by immunoblot analysis. (B) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation in HCT116 and HeLa cells. The experiments were performed as in A. (C) Effects of TRIM38 on IFNγ-induced activation of the IRF1 promoter. The experiments were performed as in A except that the IRF1 promoter reporter plasmid was used and transfected cells were treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 on TNFα- and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. HEK293 cells were transduced with either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for the indicated times, and then total RNA was prepared for qPCR analysis. Expression of TRIM38 in the stable cell lines was examined by immunoblot analysis (Right). (E) Effects of TRIM38 on TNFα- and IL-1β–induced cytokine of TNFα, IL-6, and IL-8. HEK293 cells were transduced with either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for the indicated times, and then the medium was collected for ELISA analysis. (F) Effects of TRIM38 on IFNγ-induced transcription of IRF1 gene. Cells (4 × 105) were left untreated or treated with IFNγ (100 ng/mL) for the indicated times, and total RNA was extracted for qPCR analysis. Graphs show mean ± SD; n = 3. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Over Expression, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Western Blot, Transduction, Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: Knockdown or knockout of TRIM38 potentiates TNFα- and IL-1β–triggered signaling. (A) Efficiencies of TRIM38-RNAi plasmids on TRIM38 levels. (Upper) HEK293 cells (4 × 105) were transfected with expression plasmids for TRIM38-Flag and HA-β-actin (0.1 μg each) and the indicated RNAi plasmids (1 μg each). Twenty-four hours after transfection, cell lysates were analyzed by immunoblot with anti-Flag or anti-HA. (Lower) HEK293 cells (1 × 107) were transfected with control or the indicated TRIM38-RNAi plasmids (10 μg each) for 36 h. Cell lysates were analyzed by immunoblot with anti-TRIM38 or anti–β-actin. (B) Effects of TRIM38-RNAi on TNFα- and IL-1β–triggered NF-κB activation in HEK293 and HeLa cells. The cells (1 × 105) were transfected with RNAi plasmids (1 μg each) along with the NF-κB reporter plasmid (0.01 μg). Thirty-six hours after transfection, cells were left untreated or treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for 10 h before luciferase assays were performed. (C) Effects of TRIM38-RNAi on IFNγ-induced IRF1 promoter activation. Reporter assays were performed as in B except that cells were transfected with IRF1 promoter reporter plasmid and treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 deficiency on TNFα and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. The indicated cells (4 × 105) were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for the indicated times, and then total RNA was extracted for qPCR analysis. (E) Effects of TRIM38 deficiency on TNFα and IL-1β–induced cytokine production. The indicated cells (4 × 105) were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for the indicated times, and then the medium was collected for ELISA analysis. (F) TRIM38 deficiency potentiates TNFα-triggered MAPK activation. The indicated cells (1 × 107) were left untreated or treated with TNFα (10 ng/mL) for the indicated times. Cells were lyzed and immunoblot analysis was performed with the indicated antibodies. (G) TRIM38 deficiency potentiates IL-1β–triggered MAPK activation. The experiments were performed as in E, except that cells were treated with IL-1β (10 ng/mL). Graphs show mean ± SD; n = 3. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Knockdown, Knock-Out, Transfection, Expressing, Western Blot, Control, Activation Assay, Plasmid Preparation, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: TRIM38 interacts with and destabilizes TAB2 through its C-terminal PRY-SPRY domain. (A) TRIM38 interacts with TAB2 and TAB3 in mammalian overexpression system. HEK293 cells (1 × 107) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies. (B) Endogenous TRIM38 interacts with TAB2/3. HEK293 cells (3 × 107) were left untreated or treated with TNFα (Left) or IL-1β (Right) for the indicated times. Endogenous coimmunoprecipitation and immunoblots were performed with the indicated antibodies. (C) TRIM38 specifically destabilizes TAB2/3. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h, and then immunoblots were performed with the indicated antibodies. (D) Effects of TRIM38 truncation mutants on destabilization of TAB2. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h before immunoblots were performed with the indicated antibodies. (E) Analysis of TRIM38 expression in TRIM38−/− cells stably transduced with an empty vector (II), TRIM38-Flag (III), TRIM38(63-465)-Flag (IV), or TRIM38(290-465)-Flag (V), respectively and in TRIM38+/+ cells stably transduced with an empty vector (I). Cells (1 × 107) (I, II, III, IV, V) were harvested and lysed. Immunoblot analysis was performed with the indicated antibodies. (F) Reconstitution of TRIM38 or TRIM38 mutant (63–465) into TRIM38-deficient cells leads to down-regulation of TAB2. Cells (1 × 107) (I, II, III, IV, V) were left untreated or treated with TNFα or IL-1β for the indicated times. Immunoblot analysis was performed with the indicated antibodies.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Over Expression, Transfection, Western Blot, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: TRIM38 mediates lysosomal degradation of TAB2. (A) Effects of inhibitors on TRIM38-mediated destabilization of TAB2. HEK293 cells (4 × 105) were transfected with the indicated plasmids. Fourteen hours after transfection, the cells were treated with the indicated inhibitors for 6 h before immunoblot analysis was performed. (B) Effects of NH4Cl and MG132 on down-regulation of TAB2 triggered by TNFα and IL-1β. HEK293 (1 × 107) cells were treated with NH4Cl or MG132 for 4 h and then further treated with TNFα and IL-1β for 2 h before immunoblot analysis was performed. (C) TRIM38 promotes translocation of TAB2 to the lysosome. HEK293 cells (1 × 105) were transfected with Cherry-TAB2 and GFP-LAMP1 (Left) or CFP-TRIM38 (Right). Twenty hours after transfection, cells were fixed with 4% (wt/vol) paraformaldehyde and subjected for confocal microscopy. (D) Effect of TRIM38 deficiency on TNFα- or IL-1β–induced colocalization of TAB2 with the lysosomes. TRIM38+/+, TRIM38−/−, or TRIM38−/− cells reconstituted with the PRY-SPRY domain (1 × 105) were transfected with GFP-TAB2. Twenty hours after transfection, cells were stained with Red Lysotracker (200 nM) for 2 h and treated with TNFα (20 ng/mL) or IL-1β (20 ng/mL) for 1 h and then fixed with 4% (wt/vol) paraformaldehyde and subjected to confocal microscopy. A random 10 cells in each sample were used for calculating the colocalization dots that were normalized to the total lysosome-red dots. Graphs show mean ± SD; n = 3. **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Transfection, Western Blot, Translocation Assay, Confocal Microscopy, Staining

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: RPS6KA2's role in ovarian cancer initiation, progression, prognosis, cell proliferation, and apoptosis. ( A ) Comparison of RPS6KA2 expression levels between ovarian tumor tissues (n = 426) and normal tissues (n = 88) using the GEPIA database; ( B ) immunohistochemical staining showing RPS6KA2 expression in ovarian tumor tissues (n = 3) and adjacent normal tissues (n = 3); ( C ) expression of RPS6KA2 expression across early (passages 5–20), intermediate (passages 60–80), and late (passages 120–180) passage mouse ovarian surface epithelial cells (MOSE); ( D ) t-SNE plot depicting single-cell clustering, with distinct colors representing different cell populations; ( E ) t-SNE map illustrating the spatial distribution of RPS6KA2 expression levels across individual cells, where color intensity reflects expression magnitude; ( F ) bar graph summarizing RPS6KA2 expression abundance across different cell types; ( G ) survival analysis based on the Kaplan-Meier Plotter database, evaluating the relationship of RPS6KA2 expression with progression-free survival (PFS) and overall survival (OS). Correlation analysis between RPS6KA2 mRNA levels and tumor proliferation ( H ) or apoptosis ( I ) pathway scores, with the x -axis representing RPS6KA2 expression distribution and the y -axis indicating pathway activity scores. * p < 0.05; NS, not significant ( p > 0.05)

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Comparison, Expressing, Immunohistochemical staining, Staining, Activity Assay

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: The association between RPS6KA2 and cisplatin resistance, along with the identification of its downstream target genes. ( A ) RPS6KA2 mRNA expression levels were compared between cisplatin-resistant (A2780CP and COC1/DDP) and cisplatin-sensitive cell lines (A2780 and COC1); ( B ) RPS6KA2 mRNA expression was evaluated in ovarian cancer tissues from cisplatin-resistant (n = 9) and cisplatin-sensitive (n = 9) patients; ( C ) Wilcoxon rank-sum test was used to assess the correlation between cisplatin IC 50 values and RPS6KA2 expression levels. The x -axis represents different sample groups, and the y -axis shows the distribution of IC 50 scores; ( D ) a CCK-8 assay was performed to evaluate cell viability following treatment with increasing concentrations of cisplatin, after either silencing RPS6KA2 in A2780 cells or overexpressing it in A2780CP cells; ( E ) protein–protein interaction networks involving RPS6KA2 were analyzed using the STRING database; ( F ) the correlation between PI3K-AKT-mTOR pathway activity scores and RPS6KA2 expression levels was examined. The x -axis displays the distribution of RPS6KA2 expression, and the y -axis reflects the distribution of pathway activity score; ( G ) among 188 ovarian cancer patients, stratification into low (n = 92) and high (n = 96) RPS6KA2 expression groups was conducted. Each dot represents an individual gene, with colors indicating whether predefined filtering criteria were met; ( H ) a heatmap was constructed to visualize the expression patterns of differentially expressed genes, with sample groups ordered from the outermost to the innermost layer; ( I ) KEGG pathway enrichment analysis was conducted, where color intensity indicates the level of statistical significance and circle size corresponds to the number of enriched genes (larger circles indicate more enriched genes). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Expressing, CCK-8 Assay, Activity Assay, Construct

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: Involvement of RPS6KA2 in the regulation of autophagy and cisplatin sensitivity in ovarian cancer cells. ( A ) qRT-PCR analysis was conducted to measure RPS6KA2 and autophagy-related proteins in ovarian cancer cell lines following transfection with either an RPS6KA2 overexpression plasmid or sh-RPS6KA2; ( B ) autophagic flux in A2780CP cells was evaluated using mRFP-GFP-LC3 fluorescence assay after transfection with RPS6KA2 plasmid or sh-RPS6KA2. Green signals correspond to autophagosomes, red signals indicate autolysosomes, and yellow signals reflect the early stage of autophagosome formation; ( C ) high-resolution transmission electron microscopy (×10,000 magnification) was utilized to observe ultrastructural changes associated with autophagosome formation in A2780CP cells post-transfection; ( D ) A2780 cells were categorized into five experimental groups: NC, Cisplatin (5 μg/mL), sh-RPS6KA2 + Cisplatin, sh-RPS6KA2 + TSC1 + Cisplatin, and sh-RPS6KA2 + TSC2 + Cisplatin. Cell viability was determined by CCK-8 assay (OD 450 values). ( E ) A2780CP cells were allocated into five groups: NC, Cisplatin (20 μg/mL), RPS6KA2 + Cisplatin, RPS6KA2 + sh-TSC1 + Cisplatin, and RPS6KA2 + sh-TSC2 + Cisplatin. Changes in OD 450 values were measured using the CCK-8 assay. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant ( p > 0.05)

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Quantitative RT-PCR, Transfection, Over Expression, Plasmid Preparation, Fluorescence, Transmission Assay, Electron Microscopy, CCK-8 Assay

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: Involvement of RPS6KA2 in the regulation of ferroptosis in ovarian cancer cells. ( A ) Heatmaps display the expression patterns of ferroptosis-related genes in tumor and normal tissues. The x -axis corresponds to various ferroptosis-related genes, while the y -axis reflects their relative expression levels; ( B ) Western blotting was used to examine alterations in the expression of ferroptosis-related protein expression in A2780CP cells following transfection with either RPS6KA2 overexpression plasmid or sh-RPS6KA2; ( C ) levels of intracellular Fe 2+ levels were detected by fluorescence staining (blue fluorescence marks nuclei; red fluorescence indicates Fe 2+ presence); ( D ) total iron content was quantified in A2780CP cells under different treatment conditions; ( E ) cellular GSH levels were measured as an indicator of antioxidant capacity; ( F ) changes in mitochondrial membrane potential were assessed using fluorescent probes. Green fluorescence indicates high mitochondrial membrane potential (viable cells), while red fluorescence signifies low membrane potential (apoptotic cells); Intracellular ROS accumulation was evaluated using fluorescent probes. With blue fluorescence labeling nuclei, and green fluorescence also reflecting ROS accumulation. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; NS, not significant ( p > 0.05)

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, Fluorescence, Staining, Membrane, Labeling

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: miR-512-3p enhances cisplatin resistance in ovarian cancer through direct targeting of RPS6KA2. ( A ) A comprehensive bioinformatics analysis was performed to identify upstream miRNAs that regulate RPS6KA2, using four public databases: TargetScan, miRDB, miRWalk, and StarBase databases; ( B ) expression levels of miR-512-3p were analyzed in cisplatin-resistant cell lines (A2780CP and COC1/DDP) vs cisplatin-sensitive counterparts (A2780 and COC1), as well as in clinical samples from cisplatin-resistant (n = 3) and cisplatin-sensitive (n = 3) ovarian cancer tissues; ( C ) cell viability following cisplatin exposure at different concentrations was evaluated using the CCK-8 assay after silencing miR-512-3p in A2780CP cells or overexpressing it in A2780 cells; ( D ) qRT-PCR was employed to determine alterations in RPS6KA2 expression upon transfection with either miR-512-3p mimics or inhibitor; ( E ) relative luciferase activity was measured in A2780 cells co-transfected with miR-512-3p mimics or inhibitor in combination with wild-type (wt) or mutant (mut) RPS6KA2 plasmids; ( F ) A2780 cells were grouped into four conditions: negative control (NC), cisplatin (5 μg/mL), miR-512-3p mimics + cisplatin, and miR-512-3p mimics + sh-RPS6KA2 + cisplatin. Similarly, A2780CP cells were divided into four groups: NC, cisplatin (20 μg/mL), miR-512-3p inhibitor + cisplatin, and miR-512-3p inhibitor + RPS6KA2 + cisplatin. Cell viability was assessed using the CCK-8 assay; ( G ) Apoptosis was examined by TUNEL staining in five experimental groups for each cell line. In A2780 cells: NC, cisplatin (5 μg/mL), sh-RPS6KA2 + cisplatin, miR-512-3p mimics + cisplatin, and miR-512-3p mimics + RPS6KA2 + cisplatin. In A2780CP cells: NC, cisplatin (20 μg/mL), RPS6KA2 + cisplatin, miR-512-3p inhibitor + cisplatin, and miR-512-3p inhibitor + sh-RPS6KA2 + cisplatin. Nuclei were stained blue, and apoptotic cells exhibited green fluorescence. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant ( p > 0.05)

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Expressing, CCK-8 Assay, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Mutagenesis, Negative Control, TUNEL Assay, Staining, Fluorescence

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: The miR-512-3p/RPS6KA2 axis modulates the autophagy signaling pathway. ( A ) A2780 cells were distributed into five experimental groups: negative control (NC), Cisplatin treatment (Cis), sh-RPS6KA2 + Cis, miR-512-3p mimics + Cis, and miR-512-3p mimics + Cis + RPS6KA2; ( B ) similarly, A2780CP cells were also separated into five groups: NC, cisplatin (Cis), RPS6KA2 + Cis, miR-512-3p inhibitor + Cis, and miR-512-3p inhibitor + Cis + sh-RPS6KA2. Cell immunofluorescence staining was performed to evaluate MTOR and RHEB expression levels. Nuclei were labeled in blue, MTOR signal appears in green, and RHEB is shown in red. ** p < 0.01; *** p < 0.001

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Negative Control, Immunofluorescence, Staining, Expressing, Labeling

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: The miR-512-3p/RPS6KA2 axis modulates the autophagy signaling pathway and influences cisplatin resistance in ovarian cancer. ( A ) Subcutaneous xenograft mouse models were allocated into five groups: normal control (NC), Cisplatin treatment (Cis), sh-RPS6KA2 + Cis, miR-512-3p mimics + Cis, and miR-512-3p mimics + Cis + sh-RPS6KA2. Tumor volumes were monitored and compared among groups. Immunohistochemistry analysis was performed to detect the expression levels of ATG5, ATG7, BECN1, and SQSTM1; ( B ) In a parallel experiment, subcutaneous tumor-bearing mice were divided into five groups: NC, Cis, RPS6KA2 + Cis, miR-512-3p inhibitor + Cis, and miR-512-3p inhibitor + Cis + RPS6KA2. Tumor sizes were recorded and compared. Immunohistochemistry was conducted to evaluate the expression of ATG5, ATG7, BECN1, and SQSTM1. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Control, Immunohistochemistry, Expressing

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: Targeting RPS6KA2 improves cisplatin sensitivity in ovarian cancer. A subcutaneous xenograft mouse model was established and animals were randomly assigned to 11 treatment groups receiving various interventions, including an RPS6KA2-targeted drug (Honokiol), a ferroptosis inducer (Erastin), and an autophagy inhibitor (3-MA). Tumor volumes were monitored over time and compared among the groups. Immunohistochemical analysis was also performed to evaluate alterations in the expression of RPS6KA2 and autophagy-related proteins. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: Impact of RPS6KA2 targeting on MTOR expression and apoptosis in ovarian cancer cells. Tumor-bearing mice were randomly distributed into 11 groups and administered various agents, including an RPS6KA2-targeted drug (Honokiol), a ferroptosis inducer (Erastin), and an autophagy inhibitor (3-MA). Immunofluorescence staining was performed to examine changes in MTOR expression across groups; ( A ) nuclei are labeled in blue, and MTOR signal is shown in green. ( B ) TUNEL analysis was conducted to assess apoptotic levels among groups; blue fluorescence represents the nucleus, and green fluorescence marks apoptotic cells. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant ( p > 0.05)

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Expressing, Immunofluorescence, Staining, Labeling, TUNEL Assay, Fluorescence