Journal: bioRxiv

Article Title: Cognate T-cell labeling by conventional and unconventional antigen-presenting cells in vivo using a modular SrtA-based toolkit

doi: 10.64898/2026.09.17.752294

Figure Lengend Snippet: Scheme of the LIPSTIC approach ( A ) and flow cytometry analysis ( B ) showing biotin staining in SrtA-CD40 + (D; donor) and CD40L-G5 + (A; acceptor) 291PC cells. ( C ) Intercellular labeling of CD40L-G5 + 291PC cells pre-treated or not with a blocking αCD40L or isotype antibody. Scheme of the LIPSTIC approach ( D ) and flow cytometry analysis ( E ) showing biotin staining in SrtA-hCD58 + (D) and G5-hCD2 + (A) 291PC cells. ( F ) Quantification of the LIPSTIC labeling using SrtA-hCD58/G5-hCD2 and SrtA-CD40/CD40L-G5 expressing 291PC cells. Quantification of the LIPSTIC labeling using SrtA-CD40 ( G ) or SrtA-hCD58 ( H ) and G5-hCD2 or CD40L-G5 expressing 291PCs. Mean values ± SEM of three independent experiments. Statistical significance was determined by one-way ANOVA, followed by Tukeys multiple-comparisons test. p>0.05 (ns; not significant) and p<0.01 (**).

Article Snippet: Some CD4 + T cells were incubated with an αCD40L blocking Ab (BE0017-1, Bio X Cell) or an isotype Ab (BE0091, Bio X Cell) for 30 min before the beginning of co-culture at a final concentration of 150 μg/mL.

Techniques: Flow Cytometry, Analysis, Staining, Labeling, Blocking Assay, Expressing

Journal: bioRxiv

Article Title: Fc receptor dependent and independent mechanisms of antibody-mediated enhancement of immune responses

doi: 10.64898/2026.03.27.714269

Figure Lengend Snippet: (A) Experimental schematic for B-C . (B) Plot shows the percentage of GC B cells of B220+ cells in mice pre-infused with 3BNC117 IgG2a in concert with anti-CD40L (maroon, for B-C ) or isotype control (pink, for B-C ). (C) Plot shows the percentage of PBs in mice pre-infused with 3BNC117 IgG2a in concert with anti-CD40L or isotype control. (D) Experimental schematic for E-J . (E) Plot shows the percentage of GC B cells of B220+ cells in Cr2 KO mice pre-infused with 3BNC117 IgG2a (+Ab, pink, for E-J ) or no antibody (control, -Ab, grey, for E-J ). (F) Plot shows the number of GC B cells in Cr2 KO mice pre-infused with 3BNC117 IgG2a or control. (G) Plot shows the percentage of PBs in Cr2 KO mice pre-infused with 3BNC117 IgG2a or control. (H) Plot shows the number of PBs in Cr2 KO mice pre-infused with 3BNC117 IgG2a or control. (I) Plot shows the percentage of TM4-Core+ cells of GC B cells in Cr2 KO mice pre- infused with 3BNC117 IgG2a or control. (J) Plot shows the number of TM4-Core+ GC B cells in Cr2 KO mice pre-infused with 3BNC117 IgG2a or control. Data is displayed as mean ± SD. Each point represents a single mouse from at least two independent experiments. Statistical significance for B-C was determined using two-tailed unpaired t tests with Welch’s correction. Statistical significance for E-J was determined using two-tailed Mann-Whitney U tests. ns denotes non-significant, * denotes p ≤ 0.05, ** denotes p ≤ 0.01 and **** denotes p ≤ 0.0001.

Article Snippet: Anti-CD40L (Clone: MR1, BioXCell BE0017-1) and the armenian hamster isotype control (BioXCell BE0091) were purchased from BioXCell.

Techniques: Control, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Pharmaceutical Analysis

Article Title: MST4 as a key driver of osteoclast activation in osteoporosis

doi: 10.1016/j.jpha.2025.101401

Figure Lengend Snippet: Upregulation of mammalian Sterile 20-like kinase 4 (MST4) expression in peripheral blood mononuclear cells of clinical osteoporosis patients (OP). (A) Quantitative real-time polymerase chain reaction (RT-qPCR) detection of MST4 mRNA levels in peripheral blood mononuclear cells of OP and healthy controls (Control) ( n = 6). (B, C) Western blot analysis of MST4 protein expression levels (B) and corresponding statistics (C) in OP and Control ( n = 6). (D) Bone mineral density (BMD) levels in OP and Control. (E, F) Expression levels of bone formation markers osteocalcin (OC) (E) and procollagen type I N -terminal propeptide (P1NP) (F) in OP and Control. (G, H) Expression levels of bone formation markers β-CrossLaps (β-CTX) and tartrate-resistant acid phosphatase 5b (TRAcP 5b) in OP and Control. (I) Receiver operating characteristic (ROC) curve analysis of MST4 expression for predicting fragility fractures in OP ( n = 60). ∗ P < 0.05 compared between groups, n = 60 per group（OP 60，Control 60); ns: not significant.

Article Snippet: The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Cat. No. F619534-0005, Sangon Biotech Co., Ltd., Shanghai, China), which was blocked with 5% non-fat milk (Cat. No. A600669, Sangon Biotech Co., Ltd., Shanghai, China) at room temperature for 1 h. The membrane was incubated overnight at 4 °C with primary antibodies diluted as follows: MST4 (Cat. No. ab52491, 1:100,000; Abcam, Cambridge, UK), proto-oncogene c-Fos (Cat. No. ab208942, 1:1000; c-Fos, Abcam, Cambridge, UK), nuclear factor of activated T-cells, cytoplasmic 1 (Cat. No. ab25916, 1:1000; NFATc1, Abcam, Cambridge, UK), tartrate-resistant acid phosphatase (TRAP; Cat. No. A13002, 1:1000; Abclonal, Wuhan, China), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; Cat. No. #8242, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-NF-κB (Cat. No. #3033, 1:1000;, Cell Signaling Technology, Danvers, MA, USA), mitogen-activated protein kinase (MAPK; Cat. No. #4695, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-MAPK (Cat. No. #4370, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-S/T (Cat. No. 05–1923, 1:200; Merck, Darmstadt, Germany), with GAPDH (Cat. No. ab181602, 1:10,000; Abcam, Cambridge, UK) as the internal control.

Techniques: Sterility, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Western Blot

Journal: Journal of Pharmaceutical Analysis

Article Title: MST4 as a key driver of osteoclast activation in osteoporosis

doi: 10.1016/j.jpha.2025.101401

Figure Lengend Snippet: Impact of mammalian Sterile 20-like kinase 4 (MST4) on osteoclast differentiation and resorption. (A) Outline of in vitro experimental procedures (Created by BioRender). (B, C) Assessment of MST4 knockdown and overexpression effects through quantitative real-time polymerase chain reaction (RT-qPCR) (B) and Western blot (C). (D) Representative images and statistical analysis of tartrate-resistant acid phosphatase (TRAP) staining. (E) Representative images and statistical analysis of phalloidin staining. (F) Representative images and statistical analysis of resorption pits on hydroxyapatite-coated Corning Osteo Assay plates. ∗ P < 0.05 between groups, with all cell experiments replicated three times. GAPDH: glyceraldehyde 3-phosphate dehydrogenase, RANKL: receptor activator of nuclear factor κB ligand; DAPI: 4′,6-diamidino-2-phenylindole; F-actin: filamentous actin; sh-MST4: short hairpin RNA-mediated knockdown of MST4; oe-MST4: overexpression of MST4.

Article Snippet: The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Cat. No. F619534-0005, Sangon Biotech Co., Ltd., Shanghai, China), which was blocked with 5% non-fat milk (Cat. No. A600669, Sangon Biotech Co., Ltd., Shanghai, China) at room temperature for 1 h. The membrane was incubated overnight at 4 °C with primary antibodies diluted as follows: MST4 (Cat. No. ab52491, 1:100,000; Abcam, Cambridge, UK), proto-oncogene c-Fos (Cat. No. ab208942, 1:1000; c-Fos, Abcam, Cambridge, UK), nuclear factor of activated T-cells, cytoplasmic 1 (Cat. No. ab25916, 1:1000; NFATc1, Abcam, Cambridge, UK), tartrate-resistant acid phosphatase (TRAP; Cat. No. A13002, 1:1000; Abclonal, Wuhan, China), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; Cat. No. #8242, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-NF-κB (Cat. No. #3033, 1:1000;, Cell Signaling Technology, Danvers, MA, USA), mitogen-activated protein kinase (MAPK; Cat. No. #4695, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-MAPK (Cat. No. #4370, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-S/T (Cat. No. 05–1923, 1:200; Merck, Darmstadt, Germany), with GAPDH (Cat. No. ab181602, 1:10,000; Abcam, Cambridge, UK) as the internal control.

Techniques: Sterility, In Vitro, Knockdown, Over Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, shRNA

Journal: Journal of Pharmaceutical Analysis

Article Title: MST4 as a key driver of osteoclast activation in osteoporosis

doi: 10.1016/j.jpha.2025.101401

Figure Lengend Snippet: Establishment of mouse ovariectomy (OVX) model and mammalian Sterile 20-like kinase 4 (MST4) protein expression changes. (A) Diagram of the animal experimental procedures (Created by BioRender). (B) Macroscopic morphology and weight of uteruses in Control, OVX, OVX + short hairpin RNA-mediated knockdown of MST4 (sh-MST4), and OVX + overexpression of MST4 (oe-MST4) group mice. (C) Western blot analysis of MST4 protein expression, along with bone metabolism markers proto-oncogene c-Fos (c-Fos), cytoplasmic 1 (NFATc1), and tartrate-resistant acid phosphatase (TRAP) protein expression in tibia tissues of each group of mice. ∗ P < 0.05 between groups, with 6 mice per group. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Cat. No. F619534-0005, Sangon Biotech Co., Ltd., Shanghai, China), which was blocked with 5% non-fat milk (Cat. No. A600669, Sangon Biotech Co., Ltd., Shanghai, China) at room temperature for 1 h. The membrane was incubated overnight at 4 °C with primary antibodies diluted as follows: MST4 (Cat. No. ab52491, 1:100,000; Abcam, Cambridge, UK), proto-oncogene c-Fos (Cat. No. ab208942, 1:1000; c-Fos, Abcam, Cambridge, UK), nuclear factor of activated T-cells, cytoplasmic 1 (Cat. No. ab25916, 1:1000; NFATc1, Abcam, Cambridge, UK), tartrate-resistant acid phosphatase (TRAP; Cat. No. A13002, 1:1000; Abclonal, Wuhan, China), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; Cat. No. #8242, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-NF-κB (Cat. No. #3033, 1:1000;, Cell Signaling Technology, Danvers, MA, USA), mitogen-activated protein kinase (MAPK; Cat. No. #4695, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-MAPK (Cat. No. #4370, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-S/T (Cat. No. 05–1923, 1:200; Merck, Darmstadt, Germany), with GAPDH (Cat. No. ab181602, 1:10,000; Abcam, Cambridge, UK) as the internal control.

Techniques: Sterility, Expressing, Control, shRNA, Knockdown, Over Expression, Western Blot

Journal: Journal of Pharmaceutical Analysis

Article Title: MST4 as a key driver of osteoclast activation in osteoporosis

doi: 10.1016/j.jpha.2025.101401

Figure Lengend Snippet: Influence of mammalian Sterile 20-like kinase 4 (MST4) on ovariectomy (OVX)-induced bone loss. (A) Representative micro-computed tomography (micro-CT) images of femurs in each group of mice. (B) Micro-CT analysis of bone mineral density (BMD), trabecular number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp) in each group of mice. (C) Representative images of femurs stained with hematoxylin and eosin (H&E) in each group of mice. (D, E) Representative images of femurs stained with tartrate-resistant acid phosphatase (TRAP) in each group of mice (D), along with histomorphometric analysis of osteoclast numbers per bone surface (N.Oc/BS) and osteoclast surface area per bone surface (Oc.S/BS) (E). (F) Determination of maximum force and stiffness of femurs through three-point bending test. ∗ P < 0.05 compared between the two groups, with 6 mice in each group. sh-MST4: short hairpin RNA-mediated knockdown of MST4; oe-MST4: overexpression of MST4.

Article Snippet: The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Cat. No. F619534-0005, Sangon Biotech Co., Ltd., Shanghai, China), which was blocked with 5% non-fat milk (Cat. No. A600669, Sangon Biotech Co., Ltd., Shanghai, China) at room temperature for 1 h. The membrane was incubated overnight at 4 °C with primary antibodies diluted as follows: MST4 (Cat. No. ab52491, 1:100,000; Abcam, Cambridge, UK), proto-oncogene c-Fos (Cat. No. ab208942, 1:1000; c-Fos, Abcam, Cambridge, UK), nuclear factor of activated T-cells, cytoplasmic 1 (Cat. No. ab25916, 1:1000; NFATc1, Abcam, Cambridge, UK), tartrate-resistant acid phosphatase (TRAP; Cat. No. A13002, 1:1000; Abclonal, Wuhan, China), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; Cat. No. #8242, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-NF-κB (Cat. No. #3033, 1:1000;, Cell Signaling Technology, Danvers, MA, USA), mitogen-activated protein kinase (MAPK; Cat. No. #4695, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-MAPK (Cat. No. #4370, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-S/T (Cat. No. 05–1923, 1:200; Merck, Darmstadt, Germany), with GAPDH (Cat. No. ab181602, 1:10,000; Abcam, Cambridge, UK) as the internal control.

Techniques: Sterility, Micro-CT, Staining, shRNA, Knockdown, Over Expression

Journal: Journal of Pharmaceutical Analysis

Article Title: MST4 as a key driver of osteoclast activation in osteoporosis

doi: 10.1016/j.jpha.2025.101401

Figure Lengend Snippet: Impact of tumor necrosis factor receptor-associated factor 6 (TRAF6) phosphorylation site mutations Thr463 and Thr486 on osteoclast differentiation. (A) Western blot analysis of downstream nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, osteoclast markers cellular homolog of the Finkel–Biskis–Jinkins murine osteosarcoma viral oncogene (c-Fos), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) expression levels, and statistical charts. (B) Representative images and statistical analysis of tartrate-resistant acid phosphatase (TRAP) staining. (C) Representative images and statistical analysis of tartrate-resistant acid phosphatase (TRAcP) staining. (D) Representative images and statistical analysis of resorption pits on hydroxyapatite-coated Corning Osteo Assay plate. ∗ P < 0.05 compared between groups, with all cell experiments repeated thrice. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; F-actin: filamentous actin.

Article Snippet: The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Cat. No. F619534-0005, Sangon Biotech Co., Ltd., Shanghai, China), which was blocked with 5% non-fat milk (Cat. No. A600669, Sangon Biotech Co., Ltd., Shanghai, China) at room temperature for 1 h. The membrane was incubated overnight at 4 °C with primary antibodies diluted as follows: MST4 (Cat. No. ab52491, 1:100,000; Abcam, Cambridge, UK), proto-oncogene c-Fos (Cat. No. ab208942, 1:1000; c-Fos, Abcam, Cambridge, UK), nuclear factor of activated T-cells, cytoplasmic 1 (Cat. No. ab25916, 1:1000; NFATc1, Abcam, Cambridge, UK), tartrate-resistant acid phosphatase (TRAP; Cat. No. A13002, 1:1000; Abclonal, Wuhan, China), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; Cat. No. #8242, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-NF-κB (Cat. No. #3033, 1:1000;, Cell Signaling Technology, Danvers, MA, USA), mitogen-activated protein kinase (MAPK; Cat. No. #4695, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-MAPK (Cat. No. #4370, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-S/T (Cat. No. 05–1923, 1:200; Merck, Darmstadt, Germany), with GAPDH (Cat. No. ab181602, 1:10,000; Abcam, Cambridge, UK) as the internal control.

Techniques: Phospho-proteomics, Western Blot, Protein-Protein interactions, Expressing, Staining