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: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

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: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

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: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

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: 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: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

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: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

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: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

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: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

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