Journal: Frontiers in Pharmacology

Article Title: Mycobacterium tuberculosis infection drives osteoclast overactivation via α2,3-Sialylation to promote pathological bone destruction

doi: 10.3389/fphar.2026.1738896

Figure Lengend Snippet: α2,3-sialylation is required for BCG-induced osteoclast differentiation and activity (A) Comparison of differentially expressed genes (DEGs) between BCG-infected osteoclasts (RB) and uninfected controls (R). (n = 3 biological replicates per group, differential expression was defined as |FoldChange| > 2 and padj <0.05). (B) Volcano plot of DEGs highlighting genes related to sialic acid biosynthesis. (C) KEGG pathway enrichment analysis of upregulated DEGs in RB cells. (D,E) Immunofluorescence staining of α2,3-SA in mouse calvarial sections using MAL II lectin, with quantification of α2,3-SA fluorescence intensity (n = 5). Intensity density was normalized to the PBS group mean. (F) In vitro osteoclasts subjected to MAL II lectin staining and TRAP staining, with or without sialidase treatment to enzymatically remove α2,3-SA. (G) Quantification of α2,3-SA fluorescence intensity in cultured osteoclasts (n = 5). Intensity density was measured in cellular ROIs after background subtraction and normalized to the RANKL group mean. (H) Quantification of TRAP + multinucleated cells (≥3 nuclei) per field (randomly selected fields, fixed magnification) in vitro (n = 5). (I) mRNA expression levels of osteoclast differentiation markers ( Fos, Mmp9, Nfatc1, and Ocstamp ) in osteoclasts (n = 3). Data are presented as mean ± SD. Statistical significance was determined by two-tailed unpaired Student’s t-test for two-group comparisons (E) and one-way ANOVA followed by Tukey’s post hoc test for three-group comparisons (G–I) .

Article Snippet: Biotin MAL-II , Vector laboratories , Cat# B-1265-1.

Techniques: Activity Assay, Comparison, Infection, Quantitative Proteomics, Immunofluorescence, Staining, Fluorescence, In Vitro, Cell Culture, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) SLC35A2 encodes a transporter for UDP-galactose, which is required for the synthesis of O-GalNAc glycans. B) The lectin VVA binds terminal alpha-GalNAc (Tn-antigen), while PNA binds terminal Gal of Core 1 O-GalNAc glycans (T-antigen), which can be removed by O-glycosidase (OGA). C) Breeding strategy of Emx1-Cre mice with floxed- Slc35a2 mice, with blue regions highlighting the forebrain expression Emx1 . D) VVA binding is minimal in protein blots of wild-type mouse cortex; female (+/+) and male (+/y) with floxed- Slc35a2 . In contrast, mice lacking Slc35a2 in cells expressing the forebrain-specific promoter Emx1 ( Emx1-Cre ) display robust VVA binding in both females (+/-) and males (-/y), consistent with inhibition of O-GalNAc extension. Binding of PNA after Neuraminidase A (NeuA) treatment was significantly reduced in both +/- and -/y lines, and specificity of PNA binding was confirmed via OGA treatment of a +/+ sample. ConA, which binds all N-glycans, was unaffected in all mouse lines, and specificity was confirmed using peptide N-glycosidase F (PNGase F) treatment of a +/+ sample. Each lane contains 15 µg cortical protein lysate from an individual mouse of the corresponding genotype. Experiment was replicated three times with similar results. E) Quantification of lectin binding from D normalized to total protein (TP) staining within the same lane of each blot. Data presented as the mean binding intensity +/- SEM, with data points for each individual mouse included, N = 3/genotype. One-way ANOVA confirmed significant group differences ( p < 0.05) for VVA and PNA but not ConA, followed by post hoc Student’s t-tests to confirm genotype effects in each sex: ** p -value = 0.01, **** p -value = 0.0001. F) Schematic of coronal mouse brain section highlighting the cortex (CTX), corpus callosum (CC), and basal ganglia (BG). G) VVA binding is minimal across all regions in wildtype mouse cortex (+/y), while PNA binding localizes to neuronal tracts of the CC and axon bundles in the BG. In contrast, mice lacking forebrain expression of Slc35a2 (-/y) display a build-up of truncated O-GalNAc glycans in cortex that fail to properly localize. Mature O-GalNAc glycans are entirely absent from the CTX and CC, but preserved in the BG, which does not express Emx1 -Cre. H) Relative quantification of lectin binding across brain regions (CTX, CC, BG) from G. Data presented as the mean binding intensity +/- SEM, with individual points representing the average of 6 measures across independent mice. Colored bars correspond with the specific brain region indicated in (F): CTX, purple; CC, orange; BG, blue. N = 2 for +/y VVA; N = 4 for +/- PNA, -/y VVA, and -/y PNA. Fluorescence was quantified as arbitrary intensity (a.i.) after subtracting background signal from each sample.

Article Snippet: Biotinylated lectins ConA (VectorLabs,#B-1005-5, 25 μg/mL) and VVA (VectorLabs, #B-1235-2, 25 μg/mL) were diluted in TBS-T and incubated for 1 hour at room temperature.

Techniques: Expressing, Binding Assay, Inhibition, Staining, Quantitative Proteomics, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Renal Tubule-Specific Deletion of Nephrocystin 3 (Nphp3) Causes Infantile Nephronophthisis-like Phenotypes in Mice

doi: 10.3390/ijms27062687

Figure Lengend Snippet: Renal cysts in Cdh16-Cre ; Nphp3 flox/flox mice originate from the distal tubules and collecting ducts and can be rescued by tolvaptan and 2-(2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide (CI-1040) treatment. ( A ) Immunofluorescence staining of kidneys from Cdh16-Cre ; Nphp3 flox/flox mice showing the staining of proximal tubules (LTL-FITC), distal convoluted tubules (SLC12A3-CY3), and collecting ducts (AQP2-CY5). These results indicate that renal cysts in these mice originate from the distal tubules and collecting ducts. White arrows indicate renal cysts. ( B ) Zonula occludens-1(ZO-1) immunofluorescence staining of the kidneys of 2-week-old mice, showing changes in epithelial cell polarity and disruption of epithelial integrity in the tubular walls, particularly the cyst walls, of Cdh16-Cre ; Nphp3 flox/flox mice, as indicated by the white arrows ( n = 3 mice/group). The blue fluorescence indicates cell nuclei stained with DAPI, while the green fluorescence shows the ZO-1 protein. ( C ) In 2-week-old Cdh16-Cre ; Nphp3 flox/flo x mice, renal tubular epithelial cells (particularly cyst-lining cells) exhibited a significant increase in Ki-67 positivity compared to control mice. ( D ) The gene expression levels of renal tubular injury markers (kidney injury molecule-1[KIM-1] and neutrophil gelatinase-associated lipocalin [NGAL]) were examined in 1 and 2-week-old mice. The results show that the expression of these tubular injury markers was significantly upregulated in Cre ; Nphp3 flox/flo x mice ( n = 3 mice/group). ( E ) The cyclic adenosine monophosphate (cAMP) content per unit weight of kidney tissue was measured in 1 and 2-week-old mice. The results show a certain degree of increase in cAMP levels in the kidneys of Cdh16-Cre ; Nphp3 flox/flo x mice at 1 week of age, and this increase became more pronounced and statistically significant at 2 weeks ( n ≥ 3 mice/group). ( F ) H&E-stained kidney sections (20×) from Cdh16-Cre ; Nphp3 flox/flox mice treated with tolvaptan or CI-1040. Compared with the vehicle control treatment, the administration of tolvaptan (5 mg/kg) or CI-1040 (10 mg/kg) significantly reduced renal cyst formation. In Cdh16-Cre ; Nphp3 flox/flox mice, treatment with either tolvaptan or CI-1040 significantly decreased the kidney KW/BW ratio, increased the number of glomeruli per maximal renal cross-section, and reduced the glomerular cross-sectional area ( n ≥ 3 mice/group). ( G ) Under untreated conditions, the p-ERK level in the kidneys of Cdh16-Cre ; Nphp3 flox/flox mice was significantly higher than that in Nphp3 flox/flox mice. The administration of CI-1040 (10 mg/kg) led to a significant reduction in renal phospho-ERK (p-ERK) levels in Cdh16-Cre ; Nphp3 flox/flox mice. The proportion of p-ERK-positive cells was significantly reduced following CI-1040 treatment ( n = 3 mice/group). Statistical significance was defined as * p < 0.05; *** p < 0.001; and p > 0.05 (NS [not significant]) using an unpaired two-tailed Student’s t -test for two-group comparisons.

Article Snippet: The primary antibodies used were as follows: NPHP3 (Proteintech, Cat No.: 22026-1-AP, dilution 1:200), p-ERK (Proteintech, Cat No.: 80031-1-RR, dilution 1:200), SLC12A3-CY3 (Affinity, Cat No.: AF5101, dilution 1:100, Changzhou, China), LTL-FITC (Vector Labs, Cat No.: B-1325, dilution 1:50, Newark, CA, USA), and AQP2-CY5 (Affinity, Cat No.: DF7560, dilution 1:100), CD11B-CY3 (abcam, ab133357, dilution 1:1000, Cambridge, UK), ZO-1 (Proteintech, Cat No.: 21773-1-AP, dilution 1:200), and Ki-67 (HuaBio, Cat No.: 28074-1-AP, dilution 1:200, Hangzhou, China).

Techniques: Immunofluorescence, Staining, Disruption, Fluorescence, Control, Gene Expression, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) TIM-3 expression on KASUMI-3 cells, primary AML blasts, and healthy immune subsets (CIK cells, monocytes, NK cells) assessed by flow cytometry using QuantiBRITE beads. REH (ALL cell line) served as negative control. (B) Short-term killing assay of TIM-3.CAR-CIK cells against CIK (n = 11) or KASUMI-3 (n = 8) cells compared with NT cells. Target cell lysis was evaluated by flow cytometry (E:T 5:1). (C) Short-term killing assay of TIM-3.CAR-CIK cells against monocytes (n = 8) or NK cells (n = 8) compared with NT (E:T 5:1). KASUMI-3 (n = 4) were included as positive control. (D) Immunoblot analysis of TIM-3 in lysates from monocytes, CIK cells, and KASUMI-3 cells following enzymatic treatment with PNGase F or broad neuraminidase, probed with a commercial anti–TIM-3 antibody (TIM-3-cmAb). GAPDH, loading control. Glycan symbols follow SNFG. (E) TIM-3 immunoprecipitates from monocytes, CIK cells, and KASUMI-3 cells treated with PNGase F or O- glycosidase and analyzed by immunoblot with TIM-3-cmAb and lectin far-western with Aleuria aurantia lectin (AAL; fucosylated epitopes). TGX stain-free total protein signal is shown as a loading/normalization control. (F) KASUMI-3 cells treated with vehicle (mock) or the fucosylation inhibitor 2F-peracetyl-fucose (SGN-2FF), followed by PNGase F or neuraminidase treatment and immunoblot/lectin probing with TIM-3-cmAb and AAL. See also Figure S3A . (G) Short-term killing assay of TIM-3.CAR-CIK cells against untreated or SGN-2FF-treated KASUMI-3 cells at various E:T ratios (5:1, 1:1, 0.5:1, 0.25:1 and 0.125:1, n = 8). (H) Affinity kinetics (left) and binding avidity at 1000 pN force (right) of TIM-3.CAR-CIK cells to untreated or defucosylated KASUMI-3 by LUMICKS analysis (n = 6). Immunoblot experiments (D-F) were repeated in three independent biological replicates with similar results. Data are presented as individual values and mean ± SD. Statistical significance was determined with repeated-measures two-way ANOVA with Bonferroni’s post hoc test (B, C) or using paired t test (G, H). ns, not significant; *p = 0.01, **p < 0.001, ***p = 0.0001 and ****p < 0.0001. Illustrations were created with Biorender.com. See also Figure S3 for loading-matched TIM-3 immunoprecipitation controls.

Article Snippet: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

Techniques: Expressing, Flow Cytometry, Negative Control, Lysis, Positive Control, Western Blot, Control, Glycoproteomics, Staining, Binding Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Immunoblot profiling of TIM-3 glycoforms in monocytes, CIK cells, and KASUMI-3 lysates using a recombinant scFv-derived monoclonal antibody (TIM-3scFv-mAb) following enzymatic treatment with PNGase F or broad neuraminidase. GAPDH, loading control. (B) TIM-3 immunoprecipitates from healthy monocytes, KASUMI-3 cells, and primary AML blasts treated with neuraminidase and/or PNGase F and analyzed by lectin and antibody probing: Ricinus communis agglutinin I (RCA-I; terminal β-galactose/LacNAc motifs), CA19-9 (sialyl-Lewis A), CSLEX1 (sialyl-Lewis X), and TIM-3scFv-mAb. See also Figure S3B . (C) High-resolution immunoblot of TIM-3 species detected by TIM-3scFv-mAb in CIK cells, primary AML blasts, and KASUMI-3 cells. GAPDH, loading control. See also Figure S3C . (D) RT-qPCR expression profiling of glycosyltransferases (FUT7, FUT8, ST3GAL3, ST3GAL4, ST3GAL6) in monocytes, KASUMI-3 cells, and primary AML blasts. Data are plotted as fold-change relative to monocytes and normalized to 18S RNA; individual points denote biological samples where applicable. (E) Schematic model summarizing a glycoform-biased recognition framework in which AML-associated remodeling of TIM-3 N -glycans contributes to preferential TIM-3.CAR recognition of AML-enriched TIM-3 glycoforms. Representative N -glycan structures are proposed for TIM-3 in AML blasts, monocytes and CIK cells based on enzymatic perturbation and lectin/antibody probing. Sugar moieties drawn with dashed outlines indicate features not directly resolved/assigned. Glycan symbols follow SNFG. Immunoblot and lectin/antibody blot experiments (A-C) were repeated in three independent biological replicates with similar results. Illustrations were created with Biorender.com. See also Figure S3 for additional lectin/antibody probing of TIM-3 glycoforms and terminal galactose exposure.

Article Snippet: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

Techniques: Western Blot, Recombinant, Derivative Assay, Control, Quantitative RT-PCR, Expressing, Glycoproteomics