Journal: medRxiv

Article Title: Phase Ib Study of Enzalutamide with Venetoclax in Patients with Metastatic Castration-Resistant Prostate Cancer

doi: 10.1101/2025.04.22.25326208

Figure Lengend Snippet: Venetoclax promoted BCL-2β generation and BCL-2α/BCL-2β protein degradation in both patients’ and healthy donor (HD) PBMCs. (A) Schematic of the BCL-2 genomic structure with 3 exons (top) and the mRNA and protein structures of the two major BCL-2 splice isoforms, i.e., BCL-2α and BCL-2β. Although both BCL-2α and BCL-2β have 4 BH (BCL-2 homology) domains, BCL-2α includes a C-terminal transmembrane (TM) domain needed for its anti-apoptotic function at the mitochondria whereas BCL-2β lacks this domain making it a shorter, mostly cytosolic protein lacking anti-apoptotic activity. To detect BCL-2β mRNA specifically, we designed primers targeting the unique exon-exon junction only present in the BCL-2β transcript (arrows). (B) Automated WES analysis of venetoclax-induced BCL-2β generation and BCL-2 degradation in PBMCs from 3 trial patients (Pt), who also showed PSA responses (see ). PBMCs were isolated from whole blood collected at pretreatment (Pre TX), D1 of the indicated cycles (C) or end of treatment (EoT). Note patient-specific changes in BCL-2α/β protein levels. (C) In healthy donor (HD) PBMCs, Venetoclax (ABT-199, 1 µM) caused time-dependent changes in BCL-2β mRNA levels. Briefly, the BCL-2β mRNA levels increased significantly at 2–4 h post-treatment (**p < 0.01) with a trend toward increase at 8 h but reduced at 24 h post treatment. Each dot represents an independent HD PBMC treated with ABT-199 (i.e., biological replicates) and the results were presented as fold change (FC) over the untreated samples (0 h). (D) Venetoclax (Ven) induced dynamic and donor-dependent BCL-2β and BCL-2α protein changes in HD PBMCs. In HD#1 PBMCs, venetoclax caused rapid (i.e., within 2 h) and persistent (up to 8 h) induction of BCL-2β with concomitant loss of BCL-2α leading to significant apoptosis (i.e., elevated Cl-caspase-3) such that even the loading control proteins β-actin and GAPDH were decreased/lost by 8 h. In HD#2 PBMCs, venetoclax increased BCL-2β without significant changes in BCL-2α and increased Cl-caspase within 4-8 h. In HD#3 PBMCs, venetoclax caused BCL-2β upregulation (without significant changes in BCL-2α) and increased apoptosis at around 2 h post treatment. Shown at the bottom is an image of the gel stained by Swift stain (as another loading control). Note that low levels of Cl-caspase-3 were observed in untreated (i.e., 0 h) PBMCs due to stress from the isolation process. (E) Quantification of BCL-2β protein levels (left) and BCL-2β/BCL-2α ratio as fold changes (FC; right) in HD PBMCs treated with venetoclax (i.e., ABT-199). Results represent the aggregated data from independent experiments (n=3-7) exemplified in D. (F) Western blotting showing that both BCL-2α and BCL-2β proteins were lost in HD PBMCs at β24 h post venetoclax (Ven; 1 μM) treatment.

Article Snippet: The following primary antibodies were used at 1:1000 dilution: mouse mAb against BCL-2 (Cell Signaling Technology, cat# 15071), rabbit pAb against BCL-2β (Bioss Antibodies, Woburn, MA, USA, cat# bs-15534R), rabbit pAb to cleaved CASP3 (Cell Signaling Technology, cat#9661), rabbit mAb to GAPDH (Cell Signaling Technology, cat#2118, clone 14C10), and rabbit mAb to β-actin (clone 13E5, HRP Conjugate; cat#5125).

Techniques: Activity Assay, Isolation, Control, Staining, Western Blot

Journal: The FASEB Journal

Article Title: Evidence for a fragile X messenger ribonucleoprotein 1 (FMR1) mRNA gain‐of‐function toxicity mechanism contributing to the pathogenesis of fragile X‐associated premature ovarian insufficiency

doi: 10.1096/fj.202200468rr

Figure Lengend Snippet: FIGURE 5 RP-SMS identifies proteins that bind CGG RNA aggregates in HGrC1 cells. (A) A representative distribution of H/L ratios among proteins identified in the CGG30x RNA pulldown. Results reveal that most proteins identified bind specifically to CGG30x RNA as opposed to non-specifically to beads, i.e., with 2-fold or more enrichment. (B) Venn diagram depicting the overlap between proteins identified in four replicate RP-SMS experiments, where two had ‘heavy’-labeled RNA and two had ‘light’-labeled RNA. 100 proteins are strong binders that are enriched 7-fold or more in all four experiments. (C) Western blot investigating the specificity of FUS, PA2G4 and TRA2β binding to CGG30x RNA, with beads-only and pre-let-7a-1 RNA as controls.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense antibody incubations were carried out overnight at 4°C with dilutions as follows: anti- FUS at 1:100 (AMAb90549, Atlas Antibodies), anti- PA2G4 at 1:100 (15348- 1- AP, Proteintech), anti- TRA2β at 1:400 (ab31353, Abcam), antiMSY2 at 1:1000 (Ab82527, Abcam) and anti- AMH at 1:100 (sc- 166 752, Santa Cruz).

Techniques: Labeling, Western Blot, Binding Assay

Journal: The FASEB Journal

Article Title: Evidence for a fragile X messenger ribonucleoprotein 1 (FMR1) mRNA gain‐of‐function toxicity mechanism contributing to the pathogenesis of fragile X‐associated premature ovarian insufficiency

doi: 10.1096/fj.202200468rr

Figure Lengend Snippet: FIGURE 7 Expression of FUS, PA2G4 and TRA2β in 6 month old wildtype and FXPOI mice. (A) Representative images of FUS, PA2G4 and TRA2β expression in 6 month old wildtype and CAG LoxP 5′UTR FMR1 (CGG)99x GFP x CMV Cre bigenic mice (referred to as FXPOI mice). Oocyte nuclei and cytoplasm are denoted with a red arrow head and pink asterisks, respectively, and granulosa cells, with a yellow arrow head. Scale bars represent 100 μM. (B) Quantification of mean gray values for FUS, PA2G4 and TRA2β representative of staining intensity were normalized to mean gray values of AMH (for granulosa cell data) and MSY2 (for oocyte data). Data are presented as the mean ± SEM from three separate mice. Mann–Whitney test, ****p < .0001 **p = .002.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense antibody incubations were carried out overnight at 4°C with dilutions as follows: anti- FUS at 1:100 (AMAb90549, Atlas Antibodies), anti- PA2G4 at 1:100 (15348- 1- AP, Proteintech), anti- TRA2β at 1:400 (ab31353, Abcam), antiMSY2 at 1:1000 (Ab82527, Abcam) and anti- AMH at 1:100 (sc- 166 752, Santa Cruz).

Techniques: Expressing, Staining, MANN-WHITNEY