Journal: bioRxiv

Article Title: Therapy-induced PSMA2 Sensitizes Prostate Cancer Cells to Residual Androgen and Promotes Neuroendocrine Lineage Transformation

doi: 10.1101/2025.11.25.690552

Figure Lengend Snippet: (A) Quantitative PCR graph showing fold change expression of PSMA2 mRNA normalized to GAPDH in prostate cancer cells (LNCAP or LASCPC-01 or NCI-H660) treated with enzalutamide (1uM, 48 hours) (blue bars) versus untreated controls (grey bars). For this and all subsequent graphs p-values are derived from T-test (n.s=not significant). (B) Image of a Western blot showing PSMA2 abundance in LASCPC-01 cells left untreated or treated with enzalutamide (1uM) for 48hrs. Blots were stained against PSMA2 or GAPDH as loading control. (C) Western blot image showing expression of PSMA2 in TRAMP mouse prostate tumor tissue. Twenty-five-weeks-old B6FVB TRAMP mice received regular water (Control) or water containing enzalutamide (3mg/kg) (experimental) for 6 weeks. Prostates tissues were harvested and processed for protein extraction and Western blotting. Blots were stained against PSMA2 or β-Tubulin as loading control. (D) Box plot showing PSMA2 mRNA expression in TCGA and GTEx normal (red) versus tumor (blue) prostate tissues.

Article Snippet: Blots were stained with antibodies against PSMA2 (Origene#CF505474), HSP90 (ThermoFisher Scientific # MA110372), PSA/KLK3 (Proteintech#60338-1), AR (Invitrogen # MA5-13426), and GAPDH (1:1000, DSHB-hGAPDH-2G7) or b-tubulin (1:1000, DSHB-E7).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Western Blot, Staining, Control, Protein Extraction

Journal: bioRxiv

Article Title: Therapy-induced PSMA2 Sensitizes Prostate Cancer Cells to Residual Androgen and Promotes Neuroendocrine Lineage Transformation

doi: 10.1101/2025.11.25.690552

Figure Lengend Snippet: (A-C) Quantitative PCR graphs showing the expression of SPINK1 (A) or SYP (B) or CGA (C) in PSMA2 intact or PSMA2-depleted (PSMA2-KD) LASCPC-01 cells before and after enzalutamide treatments (1uM for 48hrs). All target mRNA expression levels were normalized to GAPDH. (D-O) Representative images of intact or PSMA2-depleted (PSMA2-KD) LASCPC-01 cells left untreated or treated with enzalutamide. Cells were fixed, co-stained against Synaptophysin (Syp) and DAPI (to detect nuclei), and imaged on a confocal microscope using identical imaging parameters. Low magnification images are presented in D,G,J, and M and corresponding higher magnification images are shown E,F, H, I versus K,L,N,O) (P) Quantitative PCR graph showing the relative expression of NEPC and stemness markers ( SYP , CGA , SOX2 , PIM1 ) in LNCAP cells over-expressing PSMA2 (yellow) compared to LNCAP controls (blue). All target mRNA expression levels were normalized to GAPDH mRNA. (Q) Graph showing the amplification frequency of NEPC and stemness genes in PSMA2 amplified versus PSMA2 unaltered prostate cancer patients using the cBioportal cancer genomics analysis platform. (R) Kaplan Meier survivorship analysis of PSMA2-amplified versus unaltered prostate cancer patients from cBioportal cancer genomic analysis.

Article Snippet: Blots were stained with antibodies against PSMA2 (Origene#CF505474), HSP90 (ThermoFisher Scientific # MA110372), PSA/KLK3 (Proteintech#60338-1), AR (Invitrogen # MA5-13426), and GAPDH (1:1000, DSHB-hGAPDH-2G7) or b-tubulin (1:1000, DSHB-E7).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Microscopy, Imaging, Amplification

Journal: bioRxiv

Article Title: Therapy-induced PSMA2 Sensitizes Prostate Cancer Cells to Residual Androgen and Promotes Neuroendocrine Lineage Transformation

doi: 10.1101/2025.11.25.690552

Figure Lengend Snippet: (A) MTT assays graph showing the relative viability of control LASCPC-01 cells or PSMA2-KD LASCPC-01 cells before or after enzalutamide treatments (1um, for 48hours) treatments (red). (B) Quantitative PCR graph showing fold change expression of PSMA2 mRNA normalized to GAPDH in LASCPC-01 transfected with scramble (control) versus PSMA2 short hairpin RNA. (C) MTT assay graph showing relative viability of LASCPC-01 cells left untreated or treated with enzalutamide (1uM for 48hours) alone or together with Bortezomib (200nm for 48hours) or MG132 (10uM for 3hours). (D) MTT assay graph showing relative viability of LASCPC-01 cells left untreated or treated with R1881 (1nM) in the presence of enzalutamide (1uM) alone or together with Bortezomib (200nm). (F) Graph showing the relative proportion of live LASCPC-01 cells in FACS experiments (%propidium iodide-negative cells). LASCPC-01 cells were treated with bortezomib or MG132 or enzalutamide single or combination treatments. (F) Flank tumors from nude Foxn 1u (nu/nu) mice bearing contralateral LASCPC-01 or LASCPC-01 PSMA2 KD flank tumors (N=4; 2 males + 2 females) derived from 10 5 inoculated cells were measured once tumors reached ~15-90 mm 3 . Baseline volumes were recorded, and tumors were re-measured four days later to quantify percent change in tumor volume. Control tumors showed robust early expansion, whereas PSMA2-KD tumors displayed markedly blunted or negative growth, indicating a requirement for PSMA2 in early outgrowth. Bars represent mean SEM; significance was assessed by Welch’s t-test. (G) Kaplan Meier survivorship graph showing survival of 25-28 weeks-old B6FVB TRAMP mice left untreated or treated with Bortezomib alone or Bortezomib-enzalutamide combination treatments for two weeks.

Article Snippet: Blots were stained with antibodies against PSMA2 (Origene#CF505474), HSP90 (ThermoFisher Scientific # MA110372), PSA/KLK3 (Proteintech#60338-1), AR (Invitrogen # MA5-13426), and GAPDH (1:1000, DSHB-hGAPDH-2G7) or b-tubulin (1:1000, DSHB-E7).

Techniques: Control, Real-time Polymerase Chain Reaction, Expressing, Transfection, shRNA, MTT Assay, Derivative Assay

Journal: bioRxiv

Article Title: Therapy-induced PSMA2 Sensitizes Prostate Cancer Cells to Residual Androgen and Promotes Neuroendocrine Lineage Transformation

doi: 10.1101/2025.11.25.690552

Figure Lengend Snippet: (A) Trypan blue cell viability assay showing androgen growth response of LASCPC-01 or PSMA2-KD LASCPC-01 cells. Cells were treated with R1881 (0.05 or 0.1nM) in the absence (Untreated) or presence of enzalutamide (1uM). (B) Quantitative PCR graph showing NXK3-A expression in controls LASCPC-01 cells or PSMA2-KD LASCPC-01 cells treated with increasing levels of R1881 (0.01-1nM) in the presence of enzalutamide (1uM). Expression was normalized to GAPDH mRNA. (C and D) Western blot images from anti-AR immunoprecipitation (IP) assays on lysates derived from intact or PSMA2-depleted (PSMA2-KD) LASCPC-01 (c) or NCI-H660 (d) cells. Blots were stained with antibodies against Hsp90 or AR or β-tubulin. (E) Western blot image showing Hsp90 protein abundance in intact or PSMA2-depleted LASCPC-01 before and after treatments with increasing concentration of MG132 (10, 20, 30uM). Blots were stained with antibodies against Hsp90 or GAPDH (loading control). (F and G) Western blot image showing that direct partial over-expression of PSMA2 (F) is sufficient to sensitize LNCAP cells to androgen signaling-dependent upregulation of NKX3-1 (G). (H) Western blot images of lysates derived from controls or PSMA2-overxpressing LNCAP cells. Blots were stained against PSMA2 or or HSP90 or loading controls (β-tubulin and GAPDH, respectively). (I) MTT cell viability graph showing the relative viability of unaltered or PSMA2 over-expressing LNCAP cells following enzalutamide treatment.

Article Snippet: Blots were stained with antibodies against PSMA2 (Origene#CF505474), HSP90 (ThermoFisher Scientific # MA110372), PSA/KLK3 (Proteintech#60338-1), AR (Invitrogen # MA5-13426), and GAPDH (1:1000, DSHB-hGAPDH-2G7) or b-tubulin (1:1000, DSHB-E7).

Techniques: Viability Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunoprecipitation, Derivative Assay, Staining, Quantitative Proteomics, Concentration Assay, Control, Over Expression

Journal: Frontiers in Immunology

Article Title: NOD2 and TLR2 Signal via TBK1 and PI31 to Direct Cross-Presentation and CD8 T Cell Responses

doi: 10.3389/fimmu.2019.00958

Figure Lengend Snippet: SEC16A forms a complex with PI31 and TBK1. (A) Immunoblot analysis representing immunoprecipitation of endogenous PI31 in DCs following MDP/PAM 3 CSK 4 stimulation for LC-MS/MS analysis. (B) List of proteins from LC/MS analysis with highest confidence score and peptide counts. (C) Relative abundance protein for SEC16A. Data represent mean ± s.e.m; ( n = 2) * P < 0.05; one-way ANOVA (Tukey's multiple comparison test). (D) DCs and (E) THP1 cells stimulated with MDP/PAM 3 CSK 4 . Cell lysates were immunoprecipitated with PI31 antibodies and probed with anti-SEC16A. Data are representative of three independent experiments. Densitometric analysis of SEC16A band intensities normalized with αctin ( n = 3). Data represent the mean ± s.e.m. ( n = 3); * P < 0.05 Student's t test. (F,G) Images show confocal analysis of THP-1 cells transfected with GFP-PI31 (green) for 24 h, and stimulated with MDP/PAM 3 CSK 4 for 30 min and stained with antibody to SEC16A (red) and DAPI (blue). Pearson's coefficient for PI31 and SEC16A co-localization in control or MDP/PAM 3 CSK 4 stimulated cells. Data represent means ± s.e.m. ( n = 20), Three independent experiments with at least five images analyzed per experiment); **** P < 0.0001 Student's t -test. (H) THP1 cells were stimulated with MDP/PAM 3 CSK 4 for 30 min. Cell lysates were immunoprecipitated with anti-TBK1 antibodies and probed with anti-SEC16A, anti-TBK1 and anti-TANK antibodies. Data represent three independent experiments. Densitometric analysis of TBK1 band intensity normalized with αctin ( n = 3). Data represent the mean ± s.e.m. ( n = 3); n.s., non-significant. Student's t -test.

Article Snippet: The antibodies used in the study include: rabbit anti-human PI31 (Atlas Antibodies, HPA041122), rabbit anti-mouse PI31 (Abcam, ab187200); rabbit anti-human P38 (9212), rabbit anti-mouse/human actin (12620), anti-human TBK1 (3504), anti-human IRF-3 (11904), rabbit anti-tubulin (5335), mouse anti-human PSMB8/LMP7 (13726), rabbit anti- human PA28α (9643), rabbit anti-human PSMD2 (25430), rabbit anti-human PSMA2 (11864), rabbit anti-human TANK (2141), rabbit anti-phospho-NF-κB p65 (3033), rabbit anti-human IκBα (9242), mouse anti-human phospho-IκBα (9246), rabbit anti-mouse ERp72 (5033), rabbit anti-mouse LAMP1 (3243), rabbit anti-FLAG (14793), rabbit anti-HA (3724), rabbit anti-GFP (2956), anti-mouse (7076), and anti-rabbit (7074) IgG HRP-linked secondary antibodies were all from Cell Signaling; rabbit-anti-human SEC16A (Abcam, ab70722), rabbit anti-mouse SEC16A (Holzel diagnostika, KIAA0310), mouse anti-human PSMA2 (Enzo, BML-PW8105-0100), mouse anti-human PSMA4 (Enzo, BML-PW8120).

Techniques: Western Blot, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Transfection, Staining

Journal: Frontiers in Immunology

Article Title: NOD2 and TLR2 Signal via TBK1 and PI31 to Direct Cross-Presentation and CD8 T Cell Responses

doi: 10.3389/fimmu.2019.00958

Figure Lengend Snippet: PI31, but not SEC16A, mediates NOD2/TLR2-dependent cross-presentation. (A) PI31 expression was downregulated by transfecting BMDCs with control non-targeting and PI31 siRNAs. Immunoblots data represent three independent experiments. (B,C) 48 h post-transfection, BMDCs were incubated with MDP/PAM 3 CSK 4 in presence or absence of sOVA (0, 0.1, and 1 μg/ml) or SIINFEKL peptide for 3 h followed by co-culture with OT-I CD8 + T cells for 72 h. Cross-presentation efficiency of DCs was determined by the percentage of CD8 + CD69 + T cells using FACS. Data represent the mean ± s.e.m; ( B,C , n = 4). Data are from one experiment representative of three or more independent experiments. n.s. = non-significant; **** P < 0.0001; * P < 0.05; one-way ANOVA (Tukey's multiple comparison test) (D) SEC16A expression was downregulated by transfecting BMDCs with control non-targeting and SEC16A siRNAs. Immunoblots data represent three independent experiments. (E,F) 48 h post-transfection, BMDCs were incubated with MDP/PAM 3 CSK 4 with or without sOVA (0, 0.1, and 1 ug/ml) or SIINFEKL peptide for 3 h followed by co-culture with OT-I CD8 + T cells for 72 h. Cross-presentation was measured by calculating the percentage of CD8 − CD69 − T cells using FACS. Data represent mean ± s.e.m; ( E,F , n = 3). Data from one experiment representative of three or more independent experiments. n.s., non-significant; one-way ANOVA (Tukey's multiple comparison test).

Article Snippet: The antibodies used in the study include: rabbit anti-human PI31 (Atlas Antibodies, HPA041122), rabbit anti-mouse PI31 (Abcam, ab187200); rabbit anti-human P38 (9212), rabbit anti-mouse/human actin (12620), anti-human TBK1 (3504), anti-human IRF-3 (11904), rabbit anti-tubulin (5335), mouse anti-human PSMB8/LMP7 (13726), rabbit anti- human PA28α (9643), rabbit anti-human PSMD2 (25430), rabbit anti-human PSMA2 (11864), rabbit anti-human TANK (2141), rabbit anti-phospho-NF-κB p65 (3033), rabbit anti-human IκBα (9242), mouse anti-human phospho-IκBα (9246), rabbit anti-mouse ERp72 (5033), rabbit anti-mouse LAMP1 (3243), rabbit anti-FLAG (14793), rabbit anti-HA (3724), rabbit anti-GFP (2956), anti-mouse (7076), and anti-rabbit (7074) IgG HRP-linked secondary antibodies were all from Cell Signaling; rabbit-anti-human SEC16A (Abcam, ab70722), rabbit anti-mouse SEC16A (Holzel diagnostika, KIAA0310), mouse anti-human PSMA2 (Enzo, BML-PW8105-0100), mouse anti-human PSMA4 (Enzo, BML-PW8120).

Techniques: Expressing, Western Blot, Transfection, Incubation, Co-Culture Assay