Journal: NAR Molecular Medicine

Article Title: Identification of enzymatically modified isoquercitrin as a therapeutic lead for myotonic dystrophy type 1

doi: 10.1093/narmme/ugag007

Figure Lengend Snippet: Validation of quercetin activity in DM1 patient-derived myotubes. ( A ) RT-PCR cassette exon isoform analysis of the MBNL2 exon 5 and SYNE1 exon 137 alternative splicing events following 72 h treatment with quercetin in panel (A) DM1-A myotubes (∼1900–3000 CTG repeats) or ( B ) DM1-B myotubes (∼1300 CTG repeats) compared to independent unaffected controls “C” (mean ± SD, n = 4 biological replicates). ( C ) Cell viability assay performed on unaffected control and DM1 myotubes following 72 h treatment with quercetin at the indicated concentrations. Normalized to DMSO control (µM) (mean ± SD, n = 3 biological replicates). ( D ) RT-qPCR analysis of DMPK levels (relative to GAPDH) following quercetin treatment in DM1 and unaffected control myotubes normalized to DMSO control (0 µM) (mean ± SD, n = 3 biological replicates). ( E ) Fluorescence in situ hybridization was performed to visualize r(CUG) EXP foci using a Cy3-(CAG)8 probe, with nuclei counterstained by DAPI. Scale bar, 10 μm. (E, Left) Representative images of DM1-A and DM1-B cells treated for 72 h with either DMSO or 32 μM quercetin. (E, Right) Quantification of average number of foci per nucleus for each cell line (mean ± SD). Statistical significance is indicated. One-way ANOVA with Dunnett’s multiple comparisons test comparing treated myotubes to DMSO control (0 µM), (* P < .05, ** P < .01, *** P < .001, **** P < .0001).

Article Snippet: Samples were incubated with 1:1000 goat anti-rabbit Alexa Fluor 488 (Thermo Fisher) for 1 h at RT and mounted using VECTASHIELD Hard-Set Mounting Medium with DAPI (Vector Laboratories).

Techniques: Biomarker Discovery, Activity Assay, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Alternative Splicing, Viability Assay, Control, Quantitative RT-PCR, Fluorescence, In Situ Hybridization

Journal: NAR Molecular Medicine

Article Title: Identification of enzymatically modified isoquercitrin as a therapeutic lead for myotonic dystrophy type 1

doi: 10.1093/narmme/ugag007

Figure Lengend Snippet: Evaluating the in vivo efficacy of EMIQ in the DM1 HSA LR mouse model. ( A ) RT-qPCR analysis of HSA transgene (left panel) or endogenous Dmpk (right panel) expression (relative to Gtf2b ) in untreated HSA LR mice (UT) or following EMIQ administration in the drinking water for 6W or 12W at a low dose of 1.5 g/L (LD) or high dose of 15 g/L (HD) (mean ± SD, n = 3 biological replicates). ( B ) RT-PCR cassette exon isoform analysis of Clcn1 exon 7a exclusion event (left) and Atp2a1 exon 22 inclusion event (right) (mean ± SD, n = 3 biological replicates, ** P < .01, **** P < .0001). ( C ) HPLC measurement of tissue quercetin concentrations following EMIQ administration to WT and HSA LR mice through the drinking water. ( D ) Immunofluorescence for Clcn1 protein in untreated and in 12-week high-dose EMIQ-treated HSA LR mouse quadriceps muscle sections. Nuclei are stained using DAPI. ( E ) Measurement of myotonia grade using electromyography needle insertions in WT mice, untreated HSA LR mice, and HSA LR mice treated with high-dose EMIQ for 6 or 12 weeks (mean ± SD, n = 3 biological replicates). One-way ANOVA with Dunnett’s multiple comparisons test comparing HSA LR treatments to HSA LR untreated (* P < .05, ** P < .01, *** P < .001, **** P < .0001).

Article Snippet: Samples were incubated with 1:1000 goat anti-rabbit Alexa Fluor 488 (Thermo Fisher) for 1 h at RT and mounted using VECTASHIELD Hard-Set Mounting Medium with DAPI (Vector Laboratories).

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: Cancers

Article Title: Sialylation Inhibition Can Partially Revert Acquired Resistance to Enzalutamide in Prostate Cancer Cells

doi: 10.3390/cancers16172953

Figure Lengend Snippet: Enzalutamide-resistant prostate cancer cells have upregulation of ST6GAL1 and increased levels of α2,6-sialylated glycans. ( A ) SNA lectin immunofluorescence shows VCaP EnzR cells have increased levels of ST6GAL1 and α2-6 sialylation (SNA, the lectin from Sambucus nigra, recognises α2-6-linked sialylated N-glycans ). ( B ) SNA lectin immunofluorescence shows LNCaP EnzR cells have increased levels of ST6GAL1 and α2-6 sialylation compared to control LNCaP cells. DNA is stained with Hoechst. Scale bar = 200 µM.

Article Snippet: The cells were incubated overnight at 4 °C with FITC-conjugated SNA lectin (Vector Laboratoriesabs, FL-1301-2).

Techniques: Immunofluorescence, Control, Staining

Journal: Cancers

Article Title: Sialylation Inhibition Can Partially Revert Acquired Resistance to Enzalutamide in Prostate Cancer Cells

doi: 10.3390/cancers16172953

Figure Lengend Snippet: The sialyltransferase inhibitor P-SiaFNEtoc blocks α2,6 sialylation in the VCaP EnzR and LNCaP EnzR prostate cancer cells. ( A ) Detection of immunofluorescent staining of ST6GAL1 and α2,6-sialylation of N -glycans in the VCaP control and VCaP EnzR cells treated with 20 µM of the sialyltransferase inhibitor P-SiaFNEtoc for 6 days. Treatment of both cell lines with P-SiaFNEtoc inhibits α2,6-sialylation of N -glycans (detected using SNA lectin). Control cells were treated with DMSO. Scale bar = 50 µm. The images are representative of three biological repeats. ( B ) Detection of immunofluorescent staining of ST6GAL1 and α2,6-sialylation of N -glycans in LNCaP control and LNCaP EnzR cells treated with 2 µM of the sialyltransferase inhibitor P-SiaFNEtoc for 3 days. Treatment of both cell lines with P-SiaFNEtoc inhibits α2,6-sialylation of N -glycans (detected using SNA lectin). The control cells were treated with DMSO. Scale bar = 50 µm. The images are representative of three biological repeats.

Article Snippet: The cells were incubated overnight at 4 °C with FITC-conjugated SNA lectin (Vector Laboratoriesabs, FL-1301-2).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Phaeochromocytomas and paragangliomas harbour tumour-initiating SOX2+ stem cells

doi: 10.1101/2025.07.03.663006

Figure Lengend Snippet: In all panels, cells immunopositive for SOX2 are in brown, cells counterstained with haematoxylin. (A) Non-metastatic phaeochromocytomas PCC1 to PCC3 with mutations in NF1. (B) Non-metastatic phaeochromocytoma PCC4 with mutation in SDHC ; (C) Non- metastatic phaeochromocytomas PCC5 to PCC7 with mutations in RET (D) Malignant phaeochromocytoma PCC8 and non-malignant paraganglioma PGL9 with mutations in VHL (E) Non- malignant paragangliomas PGL12 to PGL14 and malignant paragangliomas PGL15 to PGL17 with mutations in SDHB (F) Non-malignant phaeochromocytomas PCC10 and PCC11 with no known mutations. (G) Malignant phaeochromocytomas PCC12 and PCC13 with no known mutations. Scale bars 50µm.

Article Snippet: Nuclei were counterstained with Vector Haematoxylin QS (Vector Laboratories, CAT#H-3404-100) and slides were mounted in VectaMount Permanent Mounting Medium (Vector Laboratories, CAT#H-5000-60).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Phaeochromocytomas and paragangliomas harbour tumour-initiating SOX2+ stem cells

doi: 10.1101/2025.07.03.663006

Figure Lengend Snippet: (A) Immunohistochemistry confirming the presence of SOX2+ cells in foetal adrenal medullae at 12 post-conception weeks (PCW), 17PCW and 19PCW. SOX2 positive cells in brown, tissues counterstained with haematoxylin. Scale bars 100µm. (B) In vitro isolation of foetal adrenomedullary cells at 19PCW leads to the generation of adherent colonies from 3 days under defined stem cell-promoting media. Scale bars 50µm. In vitro isolation of adult adrenomedullary cells from PCC30 benign phaeochromocytoma with no known mutations and PGL33, malignant paraganglioma with a germline mutation in SDHD , under defined stem cell-promoting media. Adherent cells and colonies seen at 24 and 96 hours. Scale bars 50µm. (C) Immunofluorescence using antibodies against SOX2 and TH confirms protein expression in PCC30 adherent colonies after two weeks in culture. Scale bars 50µm.

Article Snippet: Nuclei were counterstained with Vector Haematoxylin QS (Vector Laboratories, CAT#H-3404-100) and slides were mounted in VectaMount Permanent Mounting Medium (Vector Laboratories, CAT#H-5000-60).

Techniques: Immunohistochemistry, In Vitro, Isolation, Mutagenesis, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Phaeochromocytomas and paragangliomas harbour tumour-initiating SOX2+ stem cells

doi: 10.1101/2025.07.03.663006

Figure Lengend Snippet: (A) Single-cell suspension and labelling of foetal 19PCW cells with CellTracker green CMFDA label. Scale bars 500µm. (B) Pipeline for the in vitro isolation and in ovo transplantation of human SOX2+ cells (schematic created in BioRender.com ). (C) Chick chorioallantoic membrane (CAM) xenograft of PGL33 after 4 days of incubation. Brightfield and UV fluorescence images of the dissected CAM, with a visible cell mass established on the vascularised CAM. The silicon O-ring is visible. Red and yellow boxes indicate the magnified regions shown below, depicting the mass. (D) Immunostaining on formalin-fixed, paraffin sections of the xenograft in C. Immunofluorescence using antibodies against human nuclear antigen (HNA, red)) confirming the presence of human cells in the mass. Nuclei counterstained with DAPI. Immunohistochemistry with antibodies against SOX2 (brown), confirming the presence of SOX2-expressing cells. Nuclei counterstained with haematoxylin. Scale bars 100µm. (E) Cryosections through the lungs of chicks where CAMs were successfully grafted with either foetal adrenal stem cell cultures, isolated stem cell cultures from PCC30 or isolated stem cell cultures from PGL33. Green fluorescent cells are detected in PGL33 cultures confirming metastasis. Nuclei counterstained with DAPI. Scale bars 100µm, except on right-hand panels where scale bars are 20µm. (F) Immunofluorescence staining on cryosections through chick lungs of a PGL33 xenograft with metastasis, using antibodies against SOX2 (magenta) and TH (green). Nuclei counterstained with DAPI (blue). The greyscale images of single channels are shown for signal clarity. Yellow arrows indicate SOX2 positive cells, green arrows indicate TH positive cells and white arrows indicate SOX2;TH double-positive cells. Nuclei counterstained with DAPI. Scale bars 50µm. XenoT = xenotransplanted.

Article Snippet: Nuclei were counterstained with Vector Haematoxylin QS (Vector Laboratories, CAT#H-3404-100) and slides were mounted in VectaMount Permanent Mounting Medium (Vector Laboratories, CAT#H-5000-60).

Techniques: Suspension, In Vitro, Isolation, In Ovo, Transplantation Assay, Membrane, Incubation, Fluorescence, Immunostaining, Immunofluorescence, Immunohistochemistry, Expressing, Staining

Journal: The Journal of Biological Chemistry

Article Title: Association of Cell Surface Mucins with Galectin-3 Contributes to the Ocular Surface Epithelial Barrier *

doi: 10.1074/jbc.M109.033332

Figure Lengend Snippet: Galectin-3 localizes at the apical surface of normal epithelia in vivo and in vitro. A, confocal immunofluorescence micrograph showing binding of the rat anti-galectin-3 antibody M3/38 (green) to apical membranes of apical epithelial cells in human conjunctival (CON) and corneal (COR) tissue cross-sections. Use of the rabbit anti-galectin-3 antibody H-160 resulted in a similar staining pattern (data not shown). Propidium iodide (red) was used to visualize the cell nuclei. Magnification bars = 20 μm. B, in HCLE cells, binding of the galectin-3 antibody was predominantly apical, as evidenced along the x-y axis (en face view) and in cell culture cross-sections (inset). Magnification bar = 20 μm. C, cell surface proteins on apical cell membranes of HCLE cells were biotinylated, purified through a neutravidin-agarose affinity column, and immunoblotted with anti-galectin-3, anti-MUC1, and anti-MUC16 antibodies. Control lanes were loaded with 15 μg of total protein from HCLE cell lysates (lane 1). Biotinylation experiments were performed in duplicate, and apical proteins were detected in the bound fraction eluted from the affinity column (lanes 2 and 3). D, to determine whether biotinylation was restricted to the apical cell surface, membrane extracts of labeled cells were analyzed for the presence of integrin α5 subunit (ITGA5), a basolateral cell membrane glycoprotein in corneal epithelium (19). In these experiments ITGA5 was not detected in the bound fraction eluted from the neutravidin affinity column (lanes 2 and 3) but was detected in the unfractionated extract (lane 1) as well as in the flow-through fractions (lanes 4 and 5). E, tight junctions in HCLE cells stained with the anti-ZO-1 antibody. The image was acquired along the x-y axis and corresponds to the distribution of tight junctions observed in normal corneal epithelial cells (24). Magnification bar = 50 μm.

Article Snippet: The corresponding fluorescein isothiocyanate-conjugated secondary antibodies were applied (1:50) for 1 h at room temperature, and after a PBS wash specimens were cover-slipped using Vectashield mounting medium with propidium iodide (Vector Laboratories; Burlingame, CA).

Techniques: In Vivo, In Vitro, Immunofluorescence, Binding Assay, Staining, Cell Culture, Purification, Affinity Column, Labeling