Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Polyglutamine-Related Aggregates Can Serve as a Potent Antigen Source for Cross-Presentation by Dendritic Cells.

doi: 10.4049/jimmunol.1901535

Figure Lengend Snippet: FIGURE 3. Aggregation during apoptosis captures the steady-state proteome and di- rects MHC-I cross-presentation. (A) Western blot analysis of transfected cells express- ing Ova-RFP or RFP-Ova derivatives. Cells were treated for 12 h with 500 mM hy- droxyurea following 12 h of incubation with 100 mM etoposide. Equal numbers of cells were lysed, and sup and pellet fractions were separated on a 20% glycerol cushion and analyzed with anti-RFP and anti–lamin B1 Abs. Results are representative of three in- dependent repeats. (B) Flow cytometric analysis of 39.5 cells for GFP and RFP in- tensities representing transcription levels and abundance of Ova-RFP and RFP-Ova, re- spectively. Representative result of two bio- logical repeats. (C) ImageStream analysis of 39.5 cells before and after apoptosis induc- tion by etoposide treatment. Apoptotic cells presented were gated for AV positivity. High BDI and low area threshold indicates ag- gregated RFP. Representative cells are dis- played in (D). Results are representative of two independent experiments. (D) Rep- resentative image demonstrating RFP-Ova distribution in 39.5 cells prior and following apoptosis induction, which was confirmed using annexin 5 labeling. Results are repre- sentative of two independent repeats.

Article Snippet: Generation of EL4 and 39.5 cells expressing OVA variants Variants of Ova fused to RFP were cloned into pMIG, a retroviral expression vector (Addgene no. 9044) that harbors an IRES-GFP element.

Techniques: Western Blot, Transfection, Incubation, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Polyglutamine-Related Aggregates Can Serve as a Potent Antigen Source for Cross-Presentation by Dendritic Cells.

doi: 10.4049/jimmunol.1901535

Figure Lengend Snippet: FIGURE 4. Aggregate formation occurs in cells under- going apoptosis induction, but not in cells triggered for necrosis. (A) ImageStream analysis of 39.5 cells express- ing RFP-Ova construct, following apoptosis or necrosis induction by etoposide or heat shock, respectively. Cells were labeled with AV and DAPI and population distribu- tions were analyzed. Representative results from two in- dependent experiments. (B) Flow cytometric analysis of RFP intensity in necrotic and apoptotic AV+DAPI+ pop- ulation. (C) Representative image demonstrating RFP-Ova distribution in 39.5 cells triggered for apoptosis or ne- crosis. (D and E) ImageStream analysis of 39.5 cells in- dicating high BDI, low area threshold, and high bright field contrast (E), revealing aggregated RFP within the AV+DAPI+ gated populations. Two independent experi- ments were performed. (F) Western blot analysis of transfected cells expressing Ova-RFP upon induction of apoptosis or necrosis. Cells were lysed, sup and pellet fractions were separated on SDS-PAGE and Western blotted with anti–lamin B1 Abs. Results are representative of two independent repeats. Similar results were obtained when RFP-Ova–expressing cells were used (data not shown).

Article Snippet: Generation of EL4 and 39.5 cells expressing OVA variants Variants of Ova fused to RFP were cloned into pMIG, a retroviral expression vector (Addgene no. 9044) that harbors an IRES-GFP element.

Techniques: Construct, Labeling, Western Blot, Transfection, Expressing, SDS Page

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Polyglutamine-Related Aggregates Can Serve as a Potent Antigen Source for Cross-Presentation by Dendritic Cells.

doi: 10.4049/jimmunol.1901535

Figure Lengend Snippet: FIGURE 5. PolyQ aggregates can provide Ags for use in cross-presentation and are processed by DC. (A) Flow cytometric analysis of lymph node and spleen cells for proliferation of CD8+Va2+CD45.1+ T cell graft in mice immunized with apoptotic cells expressing RFP-Ova-RFP or 94Q-RFP-Ova-RFP. These results are representative of two independent experiments, total n = 6 (RFP-Ova-RFP) and n = 5 (94Q-RFP-Ova-RFP). ***p , 0.001. (B) In vivo killing assay. Flow cytometric analysis of spleens of immunized mice injected with SIINFEKL-loaded and control target splenocytes (CD45.1+), labeled with high CFSE (dark green) and low CFSE (light green), respectively. These data are representative of two independent experiments, total n = 6 (RFP-Ova- RFP) and n = 6 (94Q-RFP-Ova-RFP). ****p , 0.0001. (C) ImageStream analysis of 39.5 cells expressing CFP-Ova-RFP or 94Q-CFP-Ova-RFP. High BDI and high max pixel indicate aggregated RFP and CFP, whereas high max pixel and low BDI indicate diffused distribution of RFP and CFP. On right panel, the graph represents cells percentage in each gate. These results are representative of two independent experiments. (D) Representative image for CFP-Ova- RFP and 94Q-CFP-Ova-RFP cells. (E) Flow cytometric analysis of lymph node and spleen cells for proliferation (CFSE dilution) of CD8+Va2+CD45.1+ T cell graft in mice immunized with apoptotic cells expressing CFP-Ova-RFP or 94Q-CFP-Ova-RFP a week following removal of the tetracycline inducer from the media. *p , 0.05, representative of two independent experiments.

Article Snippet: Generation of EL4 and 39.5 cells expressing OVA variants Variants of Ova fused to RFP were cloned into pMIG, a retroviral expression vector (Addgene no. 9044) that harbors an IRES-GFP element.

Techniques: Expressing, In Vivo, Injection, Control, Labeling