Journal: Frontiers in Oncology

Article Title: miR-99b-5p inhibition drives apoptosis and tumor shrinkage in triple-negative breast cancer: functional characterization through AGO2-RIP-seq and mechanistic insights

doi: 10.3389/fonc.2026.1788447

Figure Lengend Snippet: Identification and functional validation of miR-99b-5p-targets involved in TRAIL-R signaling. (A) Workflow for identifying candidate miR-99b-5p target genes in MDA-MB-231 cells. RNA immunoprecipitation followed by next-generation sequencing (Ago-RIP-seq) was performed to isolate Ago-bound transcripts associated with miR-99b-5p. These transcripts were intersected with apoptosis-related genes and putative targets predicted by the miRWalk database, resulting in 65 overlapping candidates. Western blot analysis confirmed efficient and specific Ago2 immunoprecipitation, as Ago2 protein was detected in Ago2 IP samples but not in IgG controls. Pathway enrichment analysis of the 65 candidate target genes revealed significant association with the TRAIL receptor signaling pathway, which is schematically illustrated. (B) TNFRSF10B (DR5) and BAK1, key pro-apoptotic components of the TRAIL-R pathway, along with the anti-apoptotic gene BCL2L1 (BCL-XL), were selected for further analysis. Bioinformatic analysis of TNMplot RNA-seq data revealed that TNFRSF10B and BAK1 expression was reduced in tumor and metastatic breast tissues compared with normal tissues, whereas BCL2L1 expression did not differ significantly between normal and tumor samples (upper panel). Consistently, analysis of GTEx and TCGA datasets using Breast Cancer Gene-Expression Miner v5.2 showed decreased TNFRSF10B expression in breast tumors, unchanged BCL2L1 expression between normal and tumor tissues, and increased BCL2L1 expression in tumors relative to adjacent normal samples (lower panel). (C) RIP-qRT-PCR analysis demonstrating significant enrichment of TNFRSF10B transcripts in Ago immunoprecipitates from MDA-MB-231 cells transfected with miR-99b-5p mimic compared with control, indicating miR-99b-5p–dependent recruitment of DR5 mRNA to the Ago complex. (D) Western blot analysis showing that inhibition of miR-99b-5p in MDA-MB-231 cells resulted in upregulation of DR5 and BAK1 protein levels, whereas BCL-XL expression exhibited a decreasing trend without reaching statistical significance. (E) Real-time monitoring of cell proliferation using IncuCyte over 96 hours. miR-99b-5p overexpression enhanced proliferative capacity, while miR-99b-5p inhibition significantly suppressed proliferation (In the graph, navy, blue and magents colors represent miR-99b-5p-mimic, Scr and miR-99b-5p-inhibitor transfected cells respectively). (F) Luciferase reporter assays demonstrating direct binding of miR-99b-5p to the 3′ untranslated region of TNFRSF10B, validating DR5 as a direct target. Data are presented as mean ± SD from at least three independent experiments. *p<0.01 and #p<0.05 indicate statistical significance.

Article Snippet: GFP tagged anti-miR-99b-5p construct (MZIP99b-5p-PA-1), and corresponding non-target control (NTC) lentiviral construct (MZIP000-PA-1) were purchased from SBI (System Biosciences).

Techniques: Functional Assay, Biomarker Discovery, RNA Immunoprecipitation, Next-Generation Sequencing, Western Blot, Immunoprecipitation, RNA Sequencing, Expressing, Gene Expression, Quantitative RT-PCR, Transfection, Control, Inhibition, Over Expression, Luciferase, Binding Assay

Journal: Frontiers in Oncology

Article Title: miR-99b-5p inhibition drives apoptosis and tumor shrinkage in triple-negative breast cancer: functional characterization through AGO2-RIP-seq and mechanistic insights

doi: 10.3389/fonc.2026.1788447

Figure Lengend Snippet: miR-99b-5p inhibition responses of TNBC cell lines. (A) Graph showing the expression profile of miR-99b-5p in breast tumors and normal samples. TCGA data analysis showed that miR-99b-5p expression was significantly increased in breast tumors compared to normal samples (t-test, p<0.003) and it is expression is significantly higher in basal like tumors compared to other subtypes (ANOVA, p<0.05) (B) Kaplan–Meier overall survival analysis of TCGA patients stratified by miR-99b-5p expression showed that high miR-99b-5p expression is associated with poorer overall survival compared with low-expression tumors. Patients were stratified into high and low miR-99b-5p expression groups (n=541) using the median cutoff; p-values were calculated by log-rank test and HR (95% CI) was obtained from a Cox proportional hazards model (log-rank test, p=0.039). (C) Real-time viability measurements showed that downregulation of miR-99b-5p in TNBCs effectively reduced cell proliferation. (D) Representative images of apoptotic cells in scrambled control (Scr) and miR-99b-5p inhibitor–transfected TNBC cells are shown. Apoptotic rates were detected by Annexin V-FITC/PI assay and was analyzed using ACEA Novocyte flow cytometer (ACEA Biosciences Inc). Data analysis were performed with NovoExpress 1.5 (ACEA Biosciences Inc.). Bar graphs represent the apoptotic cell index. The results are shown as the mean ± SD of 3 independent biological replicates in each (**p<0.001, *p<0.05). (E) Inhibition of miR-99b-5p significantly increased G1 arrest in MDA-MB-231 and BT-20 cells, suppressing malignant cell behavior. All images represent mean of two independent experiments (*p<0.01).

Article Snippet: GFP tagged anti-miR-99b-5p construct (MZIP99b-5p-PA-1), and corresponding non-target control (NTC) lentiviral construct (MZIP000-PA-1) were purchased from SBI (System Biosciences).

Techniques: Inhibition, Expressing, Control, Transfection, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: miR-99b-5p inhibition drives apoptosis and tumor shrinkage in triple-negative breast cancer: functional characterization through AGO2-RIP-seq and mechanistic insights

doi: 10.3389/fonc.2026.1788447

Figure Lengend Snippet: Effect of miR-99b-5p on tumor growth in vivo . (A) In vivo xenograft model of miR-99b-5p inhibition. MDA-MB-231 cells stably expressing a miR-99b-5p inhibitor (LV1) or scrambled control (Scr) were subcutaneously injected into nude mice. Tumor growth was monitored for 11 weeks. LV1-derived xenografts showed significantly reduced tumor growth compared with Scr controls. Representative tumor images and H&E staining demonstrate reduced tumor mass and cellularity in LV1-derived xenografts. The left panel illustrates overall tumor architecture at low magnification (scale bar = 200 µm), whereas the right panel demonstrates cellular morphology at higher magnification (scale bar = 20 µm). qRT-PCR confirmed sustained miR-99b-5p silencing in LV1 tumors. Data are shown as mean ± SD (n = 3); *p < 0.01. (B) qRT-PCR analysis of TRAIL receptor signaling–related genes in tumors derived from LV1 or Scr cells. LV1 tumors exhibited increased expression of TNFRSF10A (DR3), TNFRSF10B (DR5), FADD, CASP8, CASP10, BID, and BAK1 compared with Scr tumors, indicating activation of the TRAIL-R signaling pathway (**p<0.0001; #p<0.01.) (C) Western blot analysis of tumor lysates showed upregulation of the pro-apoptotic receptor DR5 and downregulation of the anti-apoptotic protein BCL-XL in LV1-derived tumors relative to controls. Data are presented as mean ± SD from at least three independent experiments. *p<0.01 indicates statistical significance.

Article Snippet: GFP tagged anti-miR-99b-5p construct (MZIP99b-5p-PA-1), and corresponding non-target control (NTC) lentiviral construct (MZIP000-PA-1) were purchased from SBI (System Biosciences).

Techniques: In Vivo, Inhibition, Stable Transfection, Expressing, Control, Injection, Derivative Assay, Staining, Quantitative RT-PCR, Activation Assay, Western Blot

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: Volcano plot showing LC–MS/MS–based proteomic analysis of GFP pull-down samples from HEK293T cells expressing GFP-tagged wild-type (WT) or mutant LRRC8B. The x-axis represents log₂ fold change (mutant vs WT), and the y-axis shows −log₁₀ adjusted p-value. Proteins enriched in WT samples are shown on the left, while those enriched in mutant samples are shown on the right. Selected significantly enriched proteins are labeled. VDAC1 is identified as a WT-enriched interactor.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Mutagenesis, Labeling

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: Representative immunoblot images showing VDAC protein levels in total cell lysates and isolated mitochondrial fractions from cells transfected with GFP-tagged LRRC8B WT (W) or mutant Y380S (M) constructs. Actin (∼42 kDa) is included as a loading control for total lysates. Red boxes indicate the regions that were cropped and presented in the main figures. Molecular weight markers are shown where applicable.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Western Blot, Isolation, Transfection, Mutagenesis, Construct, Control, Molecular Weight

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: (A) Representative immunoblot images showing protein levels of GFP-tagged LRRC8B (WT and mutant) and VDAC in input lysates used for pull-down assays. (B–C) Immunoblot analysis of four independent immunoprecipitation (IP) experiments. Bands at ∼120 kDa confirm successful pull-down of GFP-tagged LRRC8B (WT and mutant) using an anti-GFP antibody. VDAC (∼32 kDa) bands indicate co-precipitation of VDAC with LRRC8B WT and mutant proteins. VDAC levels were normalized to the corresponding LRRC8B (WT or mutant) levels for quantification. Red boxes highlight the regions that were cropped and presented in the main figures. Molecular weight markers are shown where applicable.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Western Blot, Mutagenesis, Immunoprecipitation, Molecular Weight

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Time-dependent nuclear localization of Galectin-9 and electrotransfected plasmid DNA in super-resolution images. Stably Gal9-GFP–expressing C2C12 cells were electrotransfected with Cy5-labeled plasmid DNA (red) and imaged by confocal fluorescence microscopy at 3, 6, and 9 h post-ET. Hoechst (blue) was used to stain nuclei, and Gal9-GFP (green) was used to monitor Gal9 localization. Scale bars: 10 μm.

Article Snippet: This nuclear accumulation was observed not only for GFP-tagged Gal9 but also for endogenous Galectin-9, as confirmed by confocal microscopy following immunostaining ( ).

Techniques: Plasmid Preparation, Stable Transfection, Expressing, Labeling, Fluorescence, Microscopy, Staining

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Galectin-9 nuclear localization in untreated and mimosine-treated cells. (A) Quantification of cell proliferation with EdU. C2C12 cells were pretreated with or without mimosine for 24 h, followed by electrotransfection of pLuc and incubation with EdU for 16 h. The cells were analyzed with flow cytometry. The histograms of EdU signal were used to quantify the percentages of cells in G0/G1 phase, early S phase, and late S phase. (B) Typical super-resolution images of Cy5-pLuc and Gal9-GFP post-ET in untreated and mimosine-treated cells. Hoechst-stained nuclei (blue), Cy5-pDNA signals (white), colocalized Gal9-GFP with pDNA in the inner nucleus (green), and pDNA localized on the nuclear edge (red). Scale bars: 10 μm. (C) Quantification of pDNA, Gal9, and pDNA-Gal9 colocalization in untreated and mimosine-treated cells. n = 20. mean ± SEM; * P < 0.05, ** P < 0.01, ns = not significant (Mann–Whitney U test).

Article Snippet: This nuclear accumulation was observed not only for GFP-tagged Gal9 but also for endogenous Galectin-9, as confirmed by confocal microscopy following immunostaining ( ).

Techniques: Incubation, Flow Cytometry, Staining, MANN-WHITNEY

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Galectin-9 colocalizes with transcriptional nuclear hubs and accumulates in speckle-like regions following pDNA electrotransfection. (A) Representative confocal fluorescence images of C2C12 cells electrotransfected with Cy5-labeled pDNA, acquired 1 h post-electroporation. Hoechst (blue) stains the nucleus, Gal9-GFP (green) shows Gal9 localization, SC35 (red) is detected using a specific antibody, and Cy5-pDNA is shown in white. The merged image highlights areas of colocalization between Gal9 and SC35 (yellow arrow). (B) Representative confocal image showing the association of Gal9, pDNA, and SC35 at 3 h post-electroporation (left panel). The yellow arrow indicates a triple colocalization among Gal9, SC35, and pDNA. The right panel shows a super-resolution enlargement of the boxed region from the left panel. Scale bars: 10 μm.

Article Snippet: This nuclear accumulation was observed not only for GFP-tagged Gal9 but also for endogenous Galectin-9, as confirmed by confocal microscopy following immunostaining ( ).

Techniques: Fluorescence, Labeling, Electroporation

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Immunogold TEM of C2C12 nuclei following electrotransfection with or without pDNA. Immunogold TEM images of C2C12 nuclei following pDNA electrotransfection (bottom) or electrotransfection without plasmid (ET only, top), acquired 6 h post-ET. Gold particles represent Gal9 signals labeled first by a primary anti-Gal9 antibody. Scale bars: 1 μm (overview images). Boxed regions are enlarged in the corresponding panels on the right. Scale bars: 200 nm (insets).

Article Snippet: This nuclear accumulation was observed not only for GFP-tagged Gal9 but also for endogenous Galectin-9, as confirmed by confocal microscopy following immunostaining ( ).

Techniques: Plasmid Preparation, Labeling

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Time-dependent colocalization of Galectin-9 with plasmid DNA following electrotransfection and Lipofectamine-mediated transfection. (A) Quantification of Gal9-GFP and pDNA colocalization in C2C12 cells following electrotransfection. Cells transfected with pDNA were fixed at different time points (3, 6, 9, 12, 16, and 24 h) and analyzed by in-situ hybridization. (B) Quantification of Gal9-GFP and pDNA colocalization in cells transfected with Lipofectamine 3000. Cells were fixed at 3, 6, 24, 36, and 48 h post-transfection and assessed by in-situ hybridization. Data are shown as mean ± SEM ( n = 15–25, each time point); Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD test. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. (C) Representative confocal images of Gal9-GFP expressing C2C12 cells electrotransfected with Cy5-labeled nucleic acid cargos. C2C12 cells were pulsed with pDNA, minicircle DNA, mRNA, or without cargo (ET only) and imaged by confocal fluorescence microscopy at 1 h post-ET. Confocal images showing Gal9 (green), nucleic acid cargos (red), and nuclei (blue). Scale bars: 10 μm.

Article Snippet: This nuclear accumulation was observed not only for GFP-tagged Gal9 but also for endogenous Galectin-9, as confirmed by confocal microscopy following immunostaining ( ).

Techniques: Plasmid Preparation, Transfection, In Situ Hybridization, Expressing, Labeling, Fluorescence, Microscopy

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Knockdown of Galectin-9 reduces electrotransfection efficiency and pDNA transcription. (A) Western blot analysis confirming Gal9 knockdown in C2C12 cells treated with Gal9-specific siRNA compared to WT and negative siRNA (Neg) controls. β-actin was used as an internal control. (B) Representative confocal images showing nuclear distribution of Gal9, SC35, and pDNA in WT and Gal9 knockdown (KD) cells. Gal9-GFP (green), pDNA (white), SC35 (red), and nuclei stained with Hoechst (blue). The yellow arrow indicates colocalization of Gal9, pDNA, and SC35 in WT cells. Scale bars: 10 μm. (C) Quantification of electrotransfection efficiency (eTE, left) and EGFP reporter expression level (right) by flow cytometry in WT, negative control siRNA, and Gal9 siRNA knockdown groups. Data are shown as mean ± SEM ( n = 6); one-way ANOVA followed by Tukey’s HSD test was performed: **** P < 0.0001, ns, not significant.

Article Snippet: This nuclear accumulation was observed not only for GFP-tagged Gal9 but also for endogenous Galectin-9, as confirmed by confocal microscopy following immunostaining ( ).

Techniques: Knockdown, Western Blot, Control, Staining, Expressing, Flow Cytometry, Negative Control

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Overexpression of Galectin-9 enhances electrotransfection efficiency and transgene expression. (A) Western blot confirming Gal9 overexpression (OE) in C2C12 cells compared to wide type (WT) controls. β-actin served as an internal control. (B) Quantification of electrotransfection efficiency (eTE, left) and EGFP reporter expression level (right) by flow cytometry in WT and Gal9-overexpressing cells. Data are shown as mean ± SEM ( n = 4); Mann–Whitney U test: * P < 0.05.

Article Snippet: This nuclear accumulation was observed not only for GFP-tagged Gal9 but also for endogenous Galectin-9, as confirmed by confocal microscopy following immunostaining ( ).

Techniques: Over Expression, Expressing, Western Blot, Control, Flow Cytometry, MANN-WHITNEY

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Correlation between Galectin-9 expression and pDNA electrotransfection efficacy across different cell lines. (A) Western blot analysis of Gal9 protein levels in B16F10, C2C12, and 4T1 cells, with β-actin used as a control. (B) Comparison of eTE, EGFP expression level, cell viability, and apparent expression level in cells electrotransfected with an EGFP-encoding plasmid. Data are shown as mean ± SEM ( n = 3); one-way ANOVA followed by Tukey’s HSD test was performed: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, ns, not significant.

Article Snippet: This nuclear accumulation was observed not only for GFP-tagged Gal9 but also for endogenous Galectin-9, as confirmed by confocal microscopy following immunostaining ( ).

Techniques: Expressing, Western Blot, Control, Comparison, Plasmid Preparation