Journal: Bone Research

Article Title: C/EBPβ dictates postmenopausal FSHβ transcription and blockade of AEP/C/EBPβ pathway alleviates osteoporosis

doi: 10.1038/s41413-026-00510-y

Figure Lengend Snippet: #11a but not CF3CN suppresses elevation of FSH expression induced by OVX. a Ovariectomized (OVX) mice fed with vehicle, #11a or CF3CN all displayed hypoplastic thread-like uteri and atrophic ovaries, which were normal in sham mice. Uterine weight was quantified and is presented on the right. b The serum FSH levels of sham and OVX mice fed with vehicle, #11a or CF3CN (left); the serum FSH levels of ovariectomized WT and BDNF -/+ mice treated with vehicle or TrkB agonist R13 (right). n = 4–6 mice per group. c Immunoblotting of the pituitary glands from sham, OVX+ vehicle, OVX + #11a and OVX + CF3CN mice showed that #11a but not CF3CN inhibited FSHβ, pC/EBPβ, C/EBPβ and AEP levels induced by OVX. Data are representatives of three independent experiments. d Quantification of western blotting in ( c ), n = 3 per group. e The FSH levels in pituitary gland from sham and OVX mice treated with vehicle, #11a or CF3CN, detected by ELISA. n = 6 mice per group. f The RT-qPCR results of FSHβ and C/EBPβ mRNA levels in the pituitary glands from sham, OVX+ vehicle, OVX + #11a and OVX + CF3CN mice, n = 3 per group. GAPDH was employed as the internal control. Data represented as mean ± SEM, one-way ANOVA, ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Antibody to FSHβ (sc-374452, C12, 1:500 dilution for western blotting), C/EBPβ (HT-7) (catalog#: sc-7962, 1:1 000 for western blotting, 1:200 for immunofluorescence and immunohistochemistry), RANK-L (catalog#: sc-377079, 1:1 000 for western blotting), OPG (catalog#: sc-390518, 1:1 000 for western blotting), osterix (catalog#: sc-393325, 1:1 000 for western blotting) and RUNX2 (catalog#: sc-101145, 1:1 000 for western blotting) were from Santa Cruz; Antibodies to Legumain (D6S4H) (catalog#: 93627, 1:2 000 for western blotting, 1:500 for immunohistochemistry), p-C/EBPβ(catalog#: 3084 s, 1:1 000 for western blotting), Akt (catalog#:4691 s, 1:2 000 for western blotting), p-Akt(S473) (catalog#: 9271 s, 1:1 000 for western blotting), SAPK/JNK (catalog#: 9252 s, 1:1 000 for western blotting), Phospho-SAPK/JNK (Thr183/Tyr185) (catalog#: 9251 s, 1:1 000 for western blotting), ERK1/2 (9102 s, 1:1 000 dilution for western blotting), pERK1/2 (9106 s, 1:2 000 dilution for western blotting), CREB (catalog#: 9197 T, 1:1 000 for western blotting) and p-CREB (catalog#: 9198 T, 1:1 000 for western blotting) were purchased from Cell Signaling Technology; Antibody to TrkB (catalog#: MAB397, 1:1 000 for western blotting) was from R&D System; Antibodies to β-actin (catalog#: A5316, 1:3 000 for western blotting) and Fibronectin (catalog#: F3648, 1:1 000 for western blotting) were from Sigma-Aldrich; Antibody against Legumain (catalog#: AF2058-SP, 1:400 for immunofluorescence) was purchased from R&D Systems; Antibody against FSHR (PA5-50963, 1:1 000 dilution for western blotting and 1:200 for immunofluorescence) was purchased from Thermo Fisher Scientific; Antibody to MAP2 (catalog#:17490-1, 1:5 000 for immunofluorescence) was from Proteintech.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control

Journal: Nature communications

Article Title: Asthma reduces glioma formation by T cell decorin-mediated inhibition of microglia.

doi: 10.1038/s41467-021-27455-6

Figure Lengend Snippet: Fig. 3 Asthma reduces T cell induction of Ccl5 in microglia. a Schematic representation of the mouse Nf1OPG neuron-immune-cancer cell axis in the setting of asthma. b No difference in optic nerve Mdk RNA expression was observed between OVA- (n = 3) or HDM- (n = 3) treated mice and controls (n = 6). Bar graphs represent the means ± SEM of independent biological samples. One-way ANOVA with Bonferroni post hoc correction. c Ccl4 levels are similarly induced with increasing midkine concentrations (0–100 ng/ml) in CD3+ T cells from OVA- and PBS-treated mice. Data are presented as the means ± SEM of n = 3 independent biological samples. One-way ANOVA with Bonferroni post hoc correction. Data are presented as the means ± SEM. d, e Similarly increased CD8+, relative to CD4+, T cell content was detected in the optic nerves of Nf1OPG mice treated with PBS (n = 8), OVA (n = 8) or HDM (n = 8). One-way ANOVA with Bonferroni post hoc correction. Activated T cell medium from (f) OVA- (n = 4) and g HDM- (n = 4) treated mice induced lower levels of microglial Ccl5 relative to PBS-treated Nf1OPG mice. Two-tailed Student’s t test. Similar reductions of Ccl5 expression were observed in CD4+ and CD8+ T cells from (h) OVA- (n = 3) and (i) HDM- (n = 3) treated mice relative to PBS-treated controls (n = 3). One-way ANOVA with Bonferroni post hoc correction. Data are presented as the means ± SEM. d Scale bars, 40 µm. From left to right in each panel: b ns; c ns; ns; ns; ns; e P < 0.0001, P = 0.0022, P = 0.0011; f P = 0.0008; g P = 0.0015; h P = 0.0186, P = 0.0054; i P = 0.0224, P = 0.0007.

Article Snippet: In total, 5 × 105 microglia were grown in T cell-conditioned media (TCM) for 48 h8,9, followed Ccl5 (R&D Systems, MMR00) and decorin (Abcam, ab155454) determinations by ELISA.

Techniques: RNA Expression, Two Tailed Test, Expressing

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: ( A ) Recombinant human Shp2 activity after 30-min incubation with SFX-01. Data represent mean activity ( ± SEM; n = 3 biological replicates) and were fitted to a one-phase exponential decay curve (gray line; r 2 = 0.987). ( B ) Shp2 activity after incubation with SFX-01 for the indicated times and concentrations. Data shown are mean activity ( ± SEM; n = 3 biological replicates). ( C ) Shp2 activity after 30-minute incubation with or without bisphosphorylated IRS1 and SFX-01. Bar represents mean activity (± SEM; n = 4 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. ( D ) Representative immunoblots showing SFN-modification of recombinant Shp2 (1.6 nM) following incubation with 1.75 or 0.109 µM SFX-01 for 30 min. ( E ) Precursor isotopic envelop spectrum of 0.1 µM recombinant human Shp2 protein incubated with equimolar SFN for 6 h at 37 °C corresponding to a dithiolethione modification adducted between Cys 333 and Cys 367 . ( F ) Schematic representing the proposed mechanism of Shp2-dithiolethione formation by SFN. The isothiocyanate group reacts with a cysteine residue to form a dithiocarbamate intermediate, which further reacts with a second cysteine residue to yield the dithiolethione modification. ( G ) Immunoblot of immunoprecipitated WT or active site mutant Shp2 and SFN-modification from HEK cells treated with SFX-01. “E” represents non-transfected cells, and 0 h represents untreated cells. The graph represents densitometric analysis of Shp2-SFN adduct formation in WT or mutant Shp2 exposed to SFX-01 for 2 or 4 h. Bars represent mean values (± SEM; n = 3 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. .

Article Snippet: Protein-containing samples in SDS-PAGE sample buffer were subjected to electrophoresis and immunoblotting using the following primary antibodies: SFN (1:1000, in-house), Shp2 (1:1000, Abcam #32083) for recombinant protein or (R&D Systems #AF1894) for immunoprecipitation experiments, GAPDH (1:5000, CST #2118) and an anti-rabbit secondary antibody (1:2500, CST #7074).

Techniques: Recombinant, Activity Assay, Incubation, Western Blot, Modification, Residue, Immunoprecipitation, Mutagenesis, Transfection