Journal: bioRxiv

Article Title: Structural basis of NSD2 degradation via targeted recruitment of SCF-FBXO22

doi: 10.1101/2025.08.29.673087

Figure Lengend Snippet: (a) UNC10088 C -induced conformational change of the loop adjacent to C326. Structural overlay of the SCF FBXO22 -UNC10088 C -NSD2 PWWP1 model (dark gray) and the SCF FBXO22 -BACH1 BTB complex (light gray, PDB: 8UA3) , revealing the displacement of FBXO22 residues Y390. (b) Chemical structures of UNC8732 analogs that contain an aromatic aldehyde in place of the aliphatic amine of UNC8732. (c) Benzaldehyde derivatives UNC10415667 and UNC10415668 promote FBXO22-dependent ubiquitination of *NSD2 PWWP1 when added at 0.5 µM, based on fluorescent scanning of an SDS-PAGE gel. (d) Treatment of U2OS NSD2-HiBit cells with UNC10415667 (1 µM) for 6 hours resulted in a potent reduction of NSD2 levels by about 75%. A more modest effect was observed upon treatment with UNC10415668, and all other compounds had no significant effect. n ≥ 3 independent experiments. Error bars: standard error of the mean. (e) Treatment of U2OS NSD2 HiBit cells with UNC10415667 in a dose response fashion revealed a DC 50 value of 0.46 µM, which is about 2-fold more potent than UNC8732 and UNC10088. n = 3 independent experiments. Error bars: standard error of the mean. (f) UNC10415667 uses C326 to recruit NSD2 to the FBXO22 FIST domain as monitored by size-exclusion chromatography. Chromatograms of different elution profiles between mixtures of WT FBXO22 FIST or a C326A mutant with NSD2 and UNC10415667 (top). Representative Coomassie-stained SDS-PAGE gels of collected fractions. (g) Differential scanning fluorimetry (DSF) was used to monitor the stability of FBXO22 FIST wild type and variants in the presence of UNC10088 or UNC10415667 compared to DMSO, revealing that both compounds stabilize WT FBXO22 FIST and the C326 single cysteine variant (C326 only) as evidenced by an increase in melting temperature (ΔT m ), but not the C326A mutant. n = 3 independent experiments. Error bars: standard error of the mean.

Article Snippet: 9.5 μL of the master mix containing FBXO22 FIST WT or variants, SYPRO Orange Dye (Invitrogen), and DSF buffer (20 mM Tris 7.5, 150 mM NaCl, and 1 mM TCEP) were added to a 384-well qPCR plate (Genesee Scientific).

Techniques: Ubiquitin Proteomics, SDS Page, Size-exclusion Chromatography, Mutagenesis, Staining, Variant Assay

Journal: The Journal of Immunology

Article Title: In This Issue

doi: 10.4049/jimmunol.1590014

Figure Lengend Snippet: FIGURE 1. Impact of CD71+ erythroid cells on inflammatory-mediated mortality in neonates. (A) Survival after adoptive transfer with endotoxin ex- posure. Neonatal mice received 3 3 106 highly enriched neonatal CD71+ erythroid (Ter119+) cells via i.p. injection 30 min prior to endotoxin challenge (LD40). (B) Survival after adoptive transfer with polymicrobial sepsis. Neonatal mice received 5 3 106 highly enriched neonatal splenic CD71+ erythroid cells, B220+ cells, or PBS via i.p. injection 30 min prior to sepsis (LD60). (C) Gross spleen appearance and splenic CD71+ erythroid cell representation by FACS in previously healthy neonatal mice following anti-CD71 or isotype Ab treatment. Data are mean + SD. (D) Impact of anti-CD71 treatment or isotype-control Ab on sepsis survival following low (top; LD20) and high (bottom; LD60) mortality challenges. *p , 0.05, t test.

Article Snippet: Resident CD71+ erythroid splenocytes were diminished via i.p. administration of anti-CD71 mAb (150 mg/mouse; Bio X Cell, clone R17 217.1.3/TIB-219) daily for 2 d. This dose was chosen based on a previous report and our empiric results (8).

Techniques: Adoptive Transfer Assay, Injection, Control

Journal: The Journal of Immunology

Article Title: In This Issue

doi: 10.4049/jimmunol.1590014

Figure Lengend Snippet: FIGURE 3. Anti-CD71 treatment is associated with enhanced bacterial clearance due to immune priming. (A) Splenic bacterial load 24 h after nonlethal sepsis challenge (0.6 mg of cecal slurry/g body weight given 24 h after the last Ab dose) in CD71-depleted neonates that received either 5 3 106 highly enriched neonatal splenic CD71+ erythroid cells by adoptive transfer or no cells. (B) CD71 expression in healthy neonatal ileum (original magnification 3200). (C) Isotype Ab–treated neonatal ileum and anti-CD71–treated ileum 24 h after the last dose of Ab (original magnification 3200). (D) Reduction in CD71 MFI in ileum of anti-CD71–treated and isotype control–treated neonates 24 h after the last dose of Ab. (E) CFU recovered from peritoneal washes of healthy neonates 24 h after last dose of isotype Ab or anti-CD71 treatment alone. (F) Splenic bacterial load 24 h after nonlethal sepsis challenge (0.6 mg of cecal slurry/g body weight given 24 h after the last Ab dose) among neonates that received treatment with anti-CD71 Ab or isotype Ab or priming with a single dose of LPS (1 mg/g, 24 h prior to challenge). *p , 0.05, ANOVA.

Article Snippet: Resident CD71+ erythroid splenocytes were diminished via i.p. administration of anti-CD71 mAb (150 mg/mouse; Bio X Cell, clone R17 217.1.3/TIB-219) daily for 2 d. This dose was chosen based on a previous report and our empiric results (8).

Techniques: Adoptive Transfer Assay, Expressing, Control

Journal: The Journal of Immunology

Article Title: In This Issue

doi: 10.4049/jimmunol.1590014

Figure Lengend Snippet: FIGURE 4. CD71+CD235a+ cells are prominent in human neonatal cord blood and are predominantly enucleated reticulocytes. (A) Representative FACS plots for post-Ficoll mono- nuclear fraction of healthy adult peripheral blood, healthy term neonate cord blood, healthy term neonate cord blood after hypotonic lysis, enrich- ment of neonatal cord blood CD71+235a+ cells, H&E stain of enriched CD71+CD235a+ cells (top), methylene blue stain of enriched CD71+

Article Snippet: Resident CD71+ erythroid splenocytes were diminished via i.p. administration of anti-CD71 mAb (150 mg/mouse; Bio X Cell, clone R17 217.1.3/TIB-219) daily for 2 d. This dose was chosen based on a previous report and our empiric results (8).

Techniques: Lysis, Staining