Journal: bioRxiv

Article Title: ASPARAGINE-RICH METASTATIC NICHES DRIVE PROSTATE CANCER ORGANOTROPISM BY ENABLING TRANSLATIONAL REWIRING TOWARD N-GLYCOSYLATED PROTEINS

doi: 10.64898/2026.02.27.708521

Figure Lengend Snippet: (A) Western blot analysis of ASNS in metastatic cells isolated from bone (B-M-1, B-M-2) and lung (L-M-1, L-M-2) lesions. Vinculin was used as loading control. The image is representative of three independent experiments. (B) Schematic representation of asparaginyl-tRNA synthetase 1 (NARS1) mechanism of action. (C) NARS1 mRNA levels in PC3 cells following NARS1 silencing. Cells were transfected with NARS1-targeting small interfering RNA (siRNA) or negative control, and mRNA levels were evaluated after 5 days of incubation in 3D cultures (3D-C) by quantitative RT-PCR, using scramble-transfected cells as reference. (D) Relative cell number of PC3 cells silenced for NARS1 and cultured under standard 2D conditions for 5 days in the presence or absence of Asn (0.1 mM). One-way ANOVA with Sidak’s correction. (E-G) Number of putative N-glycosylation sites in proteins encoded by genes up- or down-regulated in metastatic cells derived from bone (E), lung (F), and liver (G) relative to primary tumor (PT). RNA-seq analysis was conducted as described in . Values are expressed relative to total protein number. (H-I) Fractional enrichment of UDP-GlcNAc isotopologues. PC3 cells were grown in 2D or 3D-C for 5 days and subsequently incubated in a medium containing U- 13 C-glucose for 24h. Labeling enrichment was evaluated by LC-MS analysis, and isotopologue abundance is reported as relative to total UDP-GlcNAc amount. Welch’s t test. (J) Labeling (m+5) enrichment of penotose phosphates from U- 13 C-glucose in PC3 cells growing in 2D or 3D-C for 5 days and subsequently incubated in a medium containing U- 13 C-glucose for 24h. Labeling enrichment was evaluated by LC-MS analysis. (K, L, P) Western blot analysis of GFPT1 (K), STT3a (L), and CD44 (P) expression in PC3 cells silenced for GFPT1, STT3a/b, and CD44 respectively after 48h of gene silencing. Vinculin was used as a loading control. The image is representative of three independent experiments. (M) Concanavalin A lectin binding assay performed on lysates from PC3 cells silenced or not for GFPT1 or STT3a/b and cultured in 3D-C for 5 days. Immunoblot for vinculin was used to confirm equal protein loading across samples. The image is representative of three independent experiments. (N-O) Adhesion of PC3 3D-C to collagen type I (L) and hyaluronic acid (M). Cells were cultured with Asn (0,1 mM) for 5 days and allowed to adhere for 15 min to plates coated with matrix components as reported. Adherent cells were quantified and data are shown relative to untreated cells. Welch’s t-test.

Article Snippet: Membranes were incubated for 1h at room temperature in blocking buffer (5% non-fat dry milk (SantaCruz Biotechnology #sc-2325) in PBS-Tween 0.1%), and then incubated at 4°C over-night with primary antibodies against either ATF-4 (Cell Signaling Technology #11815), ASNS (Cell Signaling Technology #92479S), Phospho-mTOR (Ser2448) (Cell Signaling Technology #5536S), mTOR (Cell Signaling Technology #2983), Phospho-S6K1(Thr389) (Cell Signalig Technology #9205), S6K1 (Cell Signaling Technology #9202), CD44 (Cell Signaling Technology #3570), GFPT1 (ABClonal #A20873), STT3a (Santa Cruz Biotechology #sc-390227), Vinculin (Santa Cruz Biotechology #sc-59803), β-Actin (Santa Cruz Biotechology #sc-47778).

Techniques: Western Blot, Isolation, Control, Transfection, Small Interfering RNA, Negative Control, Incubation, Quantitative RT-PCR, Cell Culture, Glycoproteomics, Derivative Assay, RNA Sequencing, Labeling, Liquid Chromatography with Mass Spectroscopy, Expressing, Binding Assay

Journal: bioRxiv

Article Title: ASPARAGINE-RICH METASTATIC NICHES DRIVE PROSTATE CANCER ORGANOTROPISM BY ENABLING TRANSLATIONAL REWIRING TOWARD N-GLYCOSYLATED PROTEINS

doi: 10.64898/2026.02.27.708521

Figure Lengend Snippet: (A-C) Number of putative N-glycosylation sites in proteins encoded by genes upregulated in bone-(A), lung-(B), and liver-(C) derived metastatic cells relative to the primary tumor (PT). RNA-seq analysis was conducted as described in . Values are expressed as relative to proteins number. Welch’s t-test. (D) Number of putative N-glycosylation sites in proteins encoded by genes upregulated in bone-, lung-, and liver-derived metastatic cells. One-way ANOVA with Tukey’s correction. (E) Relative incorporation of U- 13 C-glucose carbons in UDP-N-acetyl-glucosamine. PC3 cells were grown in 3D-C for 5 days and subsequently incubated in a medium containing U- 13 C-glucose for 24h. Metabolite abundance and labeling enrichment were evaluated by LC-MS analysis. (F) mRNA levels of oligosaccharyltransferase (OST) in 2D coltures and 3D-C. mRNA expression levels analyzed by quantitative RT-PCR using 2D condition as comparator. Student’s t test. (G) Concanavalin A lectin binding assay performed on lysates from PC3 cells cultured in 2D conditions or 3D-C, with or without Asn supplementation (0.1 mM). Immunoblot for actin was used to confirm equal protein loading across samples. The image is representative of three independent experiments. (H) Concanavalin A lectin binding assay performed on lysates from 3D-C cultured with or without Asn (0.1 mM) in the presence or absence of L-asparaginase (ASNase, 0.25 U/ml). Immunoblot for vinculin was used to confirm equal protein loading across samples. The image is representative of three independent experiments. (I) Total spheroid area of PC3 3D-C grown with or without Asn (0.1 mM) in the presence or absence of Tunicamycin (5ng/µl). One-way ANOVA with Dunnett’s correction. (J) Total spheroid area of Glutamine-fructose-6-phosphate transaminase 1 (GFPT1)-silenced PC3 cells grown in 3D-C with or without Asn (0.1 mM). One-way ANOVA with Dunnett’s correction. (K) Total spheroid area of 3D-C PC3 cells silenced for the OST catalytic subunits STT3A and STT3B with or without Asn (0.1 mM). One-way ANOVA with Dunnett’s correction. (L) Concanavalin A lectin binding assay performed on lysates from liver-derived (Liv-M), bone-derived (B-M-1, B-M-2) and lung-derived (L-M-1, L-M-2) metastatic cells (obtained after intracardiac or caudal artery injection, respectively) grown in 3D-C cultures. Immunoblot for vinculin was used to confirm equal protein loading across samples. The image is representative of three independent experiments. (M-P) Total spheroid area of bone-derived (M, N), and lung-derived (O, P) metastatic cells grown in 3D-C cultures with or without Asn (0.1 mM) in the presence or absence of Tunicamycin (5ng/µl). Two-way ANOVA with Tukey’s correction. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data represent mean ± s.e.m. from at least three independent experiments.

Article Snippet: Membranes were incubated for 1h at room temperature in blocking buffer (5% non-fat dry milk (SantaCruz Biotechnology #sc-2325) in PBS-Tween 0.1%), and then incubated at 4°C over-night with primary antibodies against either ATF-4 (Cell Signaling Technology #11815), ASNS (Cell Signaling Technology #92479S), Phospho-mTOR (Ser2448) (Cell Signaling Technology #5536S), mTOR (Cell Signaling Technology #2983), Phospho-S6K1(Thr389) (Cell Signalig Technology #9205), S6K1 (Cell Signaling Technology #9202), CD44 (Cell Signaling Technology #3570), GFPT1 (ABClonal #A20873), STT3a (Santa Cruz Biotechology #sc-390227), Vinculin (Santa Cruz Biotechology #sc-59803), β-Actin (Santa Cruz Biotechology #sc-47778).

Techniques: Glycoproteomics, Derivative Assay, RNA Sequencing, Incubation, Labeling, Liquid Chromatography with Mass Spectroscopy, Expressing, Quantitative RT-PCR, Binding Assay, Cell Culture, Western Blot, Injection

Journal: PLOS Pathogens

Article Title: Oligosaccharyltransferase (OST) complex inhibition effectively treats rodent and human prions

doi: 10.1371/journal.ppat.1013867

Figure Lengend Snippet: (A) Western blot showing the amplification of PK-resistant PrP Sc from brain homogenate PMCA reactions supplemented with DMSO or NGI-1. (B) Representative flow cytometry plot showing effect of siRNA knockdown of Stt3a , Stt3b , or Stt3a and Stt3b combination on surface PrP C levels in WT CAD5 cells. (C) Quantification of flow cytometry PrP C surface expression in undifferentiated CAD5 cells treated with siRNAs targeted at Stt3a , Stt3b , or Stt3a and Stt3b in combination. Points represent individual samples from biological triplicates. Asterisks represent significance values from unpaired t-tests as follows: **p 0.01, ns = not significant. (D) Western blot showing the loss of PK-resistant PrP Sc in the lysates from CAD5 cells chronically infected with 22L prions after siRNA knockdown of Stt3a , Stt3b , or Stt3a and Stt3b in combination.

Article Snippet: Western blot was performed on cell lysate to assess PK-resistant PrP Sc as described above or to assess knockdown efficiency of STT3A or STT3B using STT3A monoclonal antibody 1E2B12 (Proteintech, Rosemont, IL, USA) or STT3B polyclonal antibody (Proteintech, Cat # 15323–1-AP) as primary antibodies and horseradish peroxidase-labeled sheep-anti-mouse secondary antibody (Cytiva) or goat-anti-rabbit secondary antibody (BioRad, Hercules, CA, USA, Cat # 1706515) at manufacturers’ recommended dilutions

Techniques: Western Blot, Amplification, Flow Cytometry, Knockdown, Expressing, Infection

Journal: PLOS Pathogens

Article Title: Oligosaccharyltransferase (OST) complex inhibition effectively treats rodent and human prions

doi: 10.1371/journal.ppat.1013867

Figure Lengend Snippet: (A) Western blot showing the amplification of PK-resistant PrP Sc from brain homogenate PMCA reactions supplemented with DMSO or NGI-1. (B) Representative flow cytometry plot showing effect of siRNA knockdown of Stt3a , Stt3b , or Stt3a and Stt3b combination on surface PrP C levels in WT CAD5 cells. (C) Quantification of flow cytometry PrP C surface expression in undifferentiated CAD5 cells treated with siRNAs targeted at Stt3a , Stt3b , or Stt3a and Stt3b in combination. Points represent individual samples from biological triplicates. Asterisks represent significance values from unpaired t-tests as follows: **p 0.01, ns = not significant. (D) Western blot showing the loss of PK-resistant PrP Sc in the lysates from CAD5 cells chronically infected with 22L prions after siRNA knockdown of Stt3a , Stt3b , or Stt3a and Stt3b in combination.

Article Snippet: Western blot was performed on cell lysate to assess PK-resistant PrP Sc as described above or to assess knockdown efficiency of STT3A or STT3B using STT3A monoclonal antibody 1E2B12 (Proteintech, Rosemont, IL, USA) or STT3B polyclonal antibody (Proteintech, Cat # 15323–1-AP) as primary antibodies and horseradish peroxidase-labeled sheep-anti-mouse secondary antibody (Cytiva) or goat-anti-rabbit secondary antibody (BioRad, Hercules, CA, USA, Cat # 1706515) at manufacturers’ recommended dilutions

Techniques: Western Blot, Amplification, Flow Cytometry, Knockdown, Expressing, Infection