Journal: Science advances

Article Title: CD8 + T cell targeting of tumor antigens presented by HLA-E.

doi: 10.1126/sciadv.adm7515

Figure Lengend Snippet: Fig. 6. Stimulation of MHC-E–restricted, PAP-specific CD8+ T cells by PAP-expressing K562 cells. (A) Immunoblot of cell lysates of indicated K562-derived cell lines. Expres- sion of endogenous and transfected HLA-E in K562 cells was demonstrated with two HLA-E–specific antibodies MEM-E/02 and 3D12 (5, 90, 91). Note that nontransfected K562 cells express low amounts of HLA-E (88). V5 epitope–tagged RhPAP was detected with PAP-specific (MAB6240, R&D Systems) or V5-specific mouse monoclonal antibodies. A GAPDH-specific mAb was used as loading control. (B) ICS for IFNγ and TNFα of CD8+ T cells from three 68-1 RhCMV/RhPAP-immunized RM after coincubation with the indicated cells in the presence or absence of VL9 peptide. (C) Results from five independent experiments showing the average relative frequency (background-subtracted and normalized to K562-E/PAP) of CD69 and IFNγ and/or TNFα-positive CD8+ T cells from 68-1 RhCMV/RhPAP-immunized RM responding to the indicated cell lines in the presence or absence of VL9. Individual results are shown in data file S3. Statistical significance was determined by paired t test (not significant, P > 0.05). (D) Immunoblot for HLA-E and FLAG-tagged acid phosphatases. Lysates of stable cell lines of AA7 cells (= K562 cells deleted for HLA-E; fig. S7) transfected with HLA-E alone or together with human PAP, ACP2, or ACPT were electrophoretically separated and probed with the indicated antibodies by immunoblot. (E) ICS for IFNγ and TNFα of CD8+ T cells from three 68-1 RhCMV/RhPAP-immunized RM after coincubation with the indicated cell lines. (F) Average frequencies (+SEM) of CD69 and IFNγ and/or TNFα-positive CD8+ T cells responding to the indicated cell lines were background subtracted and normalized to AA7-E/PAP. Individual results are shown in data file S3. Statistical significance was determined using paired t test.

Article Snippet: Following blocking with 5% milk in PBS–0.1% Tween- 20 (Thermo Fisher Scientific) (PBS- T), membranes were probed with the following antibodies in 5% milk in PBS- T: anti–MHC- E 3D12 (1:1000; Invitrogen 14- 9953- 82), anti–MHC- E MEM- E/02 (1:5000; Invitrogen MA1- 19304), antiFLAG epitope (1:5000; Sigma- Aldrich #F3165), anti- V5 epitope (1:500; Invitrogen #37- 7500), anti–glyceraldehyde phosphate dehydrogenase (GAPDH; 1:5000; Invitrogen #MA5- 15738), anti- HA epitope (1:2000; clone HA- 7, Sigma- Aldrich H9658), anti- PAP (1:500; MAB6240, R&D Systems) anti–MHC- I heavy chain (1:5000; HC10, Thermo Fisher Scientific), or anti- RhCMV IE2 (15 μg/ml; clone IIA5.2) (53).

Techniques: Expressing, Western Blot, Derivative Assay, Transfection, Bioprocessing, Control, Stable Transfection

Journal: Science advances

Article Title: CD8 + T cell targeting of tumor antigens presented by HLA-E.

doi: 10.1126/sciadv.adm7515

Figure Lengend Snippet: Fig. 7. Recognition of PCa cell lines by MHC-E–restricted, PAP-specific CD8+ T cells. (A) Immunoblot of cell lysates of the indicated cell lines for HLA-E, PAP, HLA-Ia, and GAPDH. Antibodies used were MEM-E/02 for HLA-E (90), HC10 for HLA-Ia heavy chains (92), and MA5-15738 (Invitrogen) for GAPDH. The anti-PAP antibody (MAB6240 R&D Systems) detected a lower molecular weight band in DU145 cells that is likely nonspecific since DU145 cells are known to be PAP negative (93, 94). (B) ICS for IFNγ and TNFα production of CD8+ T cells from a 68-1 RhCMV/RhPAP-immunized RM after coincubation with the indicated cell lines in the presence or absence of VL9 peptide. In addition, we inhibited potential MHC-II presentation by adding anti-DR and CLIP peptide to all stimulations. (C) Average frequencies (+SEM) of CD69 and IFNγ and/or TNFα-positive CD8+ T cells from 68-1 RhCMV/RhPAP-immunized RM after background subtraction. The number of repeat experiments is indicated. Individual results are shown in data file S3. Statistical significance was calculated using paired Mann-Whitney U test (not significant, P > 0.05).

Article Snippet: Following blocking with 5% milk in PBS–0.1% Tween- 20 (Thermo Fisher Scientific) (PBS- T), membranes were probed with the following antibodies in 5% milk in PBS- T: anti–MHC- E 3D12 (1:1000; Invitrogen 14- 9953- 82), anti–MHC- E MEM- E/02 (1:5000; Invitrogen MA1- 19304), antiFLAG epitope (1:5000; Sigma- Aldrich #F3165), anti- V5 epitope (1:500; Invitrogen #37- 7500), anti–glyceraldehyde phosphate dehydrogenase (GAPDH; 1:5000; Invitrogen #MA5- 15738), anti- HA epitope (1:2000; clone HA- 7, Sigma- Aldrich H9658), anti- PAP (1:500; MAB6240, R&D Systems) anti–MHC- I heavy chain (1:5000; HC10, Thermo Fisher Scientific), or anti- RhCMV IE2 (15 μg/ml; clone IIA5.2) (53).

Techniques: Western Blot, Molecular Weight, MANN-WHITNEY

Journal: Science advances

Article Title: CD8 + T cell targeting of tumor antigens presented by HLA-E.

doi: 10.1126/sciadv.adm7515

Figure Lengend Snippet: Fig. 8. Recognition of primary PCa cells by MHC-E–restricted, PAP-specific CD8+ T cells. Left: ICS for IFNγ and TNFα production by CD8+ T cells from two 68-1 RhCMV/ RhPAP-immunized RM after coincubation with PCa cell suspensions in the presence or absence of VL9 peptide. In addition, we inhibited MHC-II presentation by adding anti-DR antibody and CLIP peptide in all stimulations. Right: Summary of results from the indicated number of primary PCa samples showing the average frequency of CD69 and IFNγ and/or TNFα-positive CD8+ T cells from 68-1 RhCMV/RhPAP-immunized RM in the absence or presence of VL9 peptide after background subtraction. Indi- vidual results are shown in data file S3. Statistical significance was calculated using paired t test.

Article Snippet: Following blocking with 5% milk in PBS–0.1% Tween- 20 (Thermo Fisher Scientific) (PBS- T), membranes were probed with the following antibodies in 5% milk in PBS- T: anti–MHC- E 3D12 (1:1000; Invitrogen 14- 9953- 82), anti–MHC- E MEM- E/02 (1:5000; Invitrogen MA1- 19304), antiFLAG epitope (1:5000; Sigma- Aldrich #F3165), anti- V5 epitope (1:500; Invitrogen #37- 7500), anti–glyceraldehyde phosphate dehydrogenase (GAPDH; 1:5000; Invitrogen #MA5- 15738), anti- HA epitope (1:2000; clone HA- 7, Sigma- Aldrich H9658), anti- PAP (1:500; MAB6240, R&D Systems) anti–MHC- I heavy chain (1:5000; HC10, Thermo Fisher Scientific), or anti- RhCMV IE2 (15 μg/ml; clone IIA5.2) (53).

Techniques:

Journal: EMBO Molecular Medicine

Article Title: Targeting pre-existing club-like cells in prostate cancer potentiates androgen deprivation therapy

doi: 10.1038/s44321-026-00375-y

Figure Lengend Snippet: ( A , B ) Expression per Pten pc−/− LSC med cell subpopulation ( A ) and UMAP projection ( B ) of Pim1 . ( C , D ) Number ( C ) and size ( D ) of organoids generated from sorted Pten pc−/− LSC med cells after 10 days of culture in medium containing DMSO or 1 nM JQ-1 and 1 nM CX-6258, alone or combined, as indicated. Data were normalized to the control (DMSO) condition (biological replicates, n = 5 independent experiments). ** p < 0.01; *** p < 0.001; **** p < 0.0001 (one-way ANOVA and Dunnett post hoc test). Exact p values are reported in Appendix Table . ( E , F ) Enrichment of various epithelial cell signatures ( E ) and LSC med subpopulation signatures ( F ) in HPV-10, PC-3, DU145, LNCaP, and 22Rv1 cell lines (Data Ref: Wang et al, ). ( G ) Enrichment of CRPC-AR, CRPC-WNT, CRPC-NE, and CRPC-SCL human tumoral subtypes signatures (Dataset ) in HPV-10, PC-3, DU145, LNCaP, and 22Rv1 cell lines. ( H ) Human HPV-10 cells were treated for 72 h with 1 µM JQ-1 and 1 µM CX-6258, alone or combined (as indicated), then the number of adherent cells was counted (biological replicates, n = 4 independent experiments). The data were normalized to the control condition (DMSO). ** p < 0.01; *** p < 0.001 (one-way ANOVA and Dunnett post hoc test). Exact p values are reported in Appendix Table . ( I ) Images of HPV-10 cells after 72 h of treatment. ( J ) The viability of HPV-10 cells (adherent + in suspension) was determined by trypan blue staining (biological replicates, n = 4 independent experiments). ns not significant; * p < 0.05 (one-way ANOVA and Dunnett post hoc test). Exact p values are reported in Appendix Table . ( K , L ) Same as ( H , J ) with PC-3 cells (biological replicates, n = 4 independent experiments). ns not significant; * p < 0.05, ** p < 0.01; *** p < 0.001 (one-way ANOVA and Dunnett post hoc test). Exact p values are reported in Appendix Table . ( M ) Tumorsphere-forming capacity of HPV-10 cells in medium containing DMSO or 1 µM JQ-1 and 1 µM CX - 6258, alone or combined as indicated. Data were normalized to the DMSO condition (biological replicates, n = 3 independent experiments). *** p < 0.001 (one-way ANOVA and Dunnett post hoc test). Exact p values are reported in Appendix Table . All error bars in this figure represent SD. .

Article Snippet: HPV-10 , ATCC , CRL-2220 RRID: CVCL_3495.

Techniques: Expressing, Generated, Control, Suspension, Staining

Journal: EMBO Molecular Medicine

Article Title: Targeting pre-existing club-like cells in prostate cancer potentiates androgen deprivation therapy

doi: 10.1038/s44321-026-00375-y

Figure Lengend Snippet: ( A , B ) Dose-response of JQ-1 ( A ) and CX-6258 ( B ) on the number of organoids formed by sorted Pten pc−/− LSC med cells. Data are normalized to the DMSO condition (biological replicates, n = 5 ( A ) and n = 4 ( B ) independent experiments). **** p < 0.0001 versus DMSO (ANOVA analysis and Dunnett’s post hoc test). Exact p values are reported in Appendix Table . ( C ) Expression of FOSL1 and PIM1 in human HPV-10 cells determined by RT-qPCR. The results are normalized to the values obtained in LNCaP cells, represented by the horizontal dotted line (biological replicates, n = 3 independent experiments). * p < 0.05 versus LNCaP cells (unpaired t -test with Welch’s correction), p = 0.0157 ( FOSL1 ), p = 0.0278 ( PIM1 ). ( D ) Dose-response of JQ-1 and CX-6258 on the number of adherent HPV-10 cells. Data were normalized to the DMSO condition (each dot is the average of three biological replicates). **** p < 0.0001 versus DMSO (ordinary two-way ANOVA with Šídák multiple comparisons test). Exact p values are reported in Appendix Table . ( E – H ) Human PC-3 ( E , F ) and HPV-10 ( G , H ) cells were treated with T5224-PROTAC (‘PROTAC’) at 0, 8, and 24 h. The cells were collected at 48 h. The cell number ( E , G ) and cell viability (adherent + in suspension) ( F , H ) were determined by trypan blue staining (biological replicates, n = 4 independent experiments). The data were normalized to DMSO (control condition). * p < 0.05, ** p < 0.01 (ANOVA analysis and Dunn’s post hoc test). ( I ) Human PC-3 cells were treated with siRNA (siScrambled or three different FOSL1 siRNA, as indicated) for 6 h and the cells were collected at 48 h. The expression of FOSL1 was measured by RT-qPCR (biological replicates, n = 2 independent experiments). The data were normalized to siScrambled (control condition). The three siRNA FOSL1 showed similar efficacy. **** p < 0.0001 (ANOVA analysis and Dunnett’s post hoc test). Exact p values are reported in Appendix Table . ( J – L ) Human PC-3 cells were treated with siScrambled or siFOSL1(1) for 6 h and the cells were collected at 72 h. Cell number ( J ) and cell viability (adherent + in suspension) ( K ) were determined by trypan blue staining. The expression of FOSL1 ( L ) was measured by RT-qPCR. The data are normalized to siScrambled (biological replicates, n = 3 independent experiments). ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-tailed t -test). Exact p values are reported in Appendix Table . All error bars in this figure represent SD.

Article Snippet: HPV-10 , ATCC , CRL-2220 RRID: CVCL_3495.

Techniques: Expressing, Quantitative RT-PCR, Suspension, Staining, Control, Two Tailed Test