Journal: Frontiers in Immunology

Article Title: Next-generation cell lines for profiling different proteasome forms and their implications in cancer

doi: 10.3389/fimmu.2025.1672000

Figure Lengend Snippet: Generation of SW620B8-mCherryB5-GFP and TZM-blB8-mCherryB10-CFP cell lines. (A) Schematic representation of genome modifications in SW620B8-mCherryB5-GFP (left) and TZM-blB8-mCherryB10-CFP (right) cell lines. (B) Analysis of SW620- and TZM-bl-derived cells by flow cytometry. Populations of control cells and cells stimulated for 72 h with pro-inflammatory cytokines (1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF) are shown. Control cell populations are shown in red; cells expressing one chimeric subunit – in blue, and cells with two chimeric subunits – in orange. For each cell population, FSC vs SSC gating was used to exclude dead cells and cell debris; FSC area vs height gating was done to exclude cell doublets. 10.000 events are shown in each case. (C) Mean fluorescence intensity of mCherry, EGFP and PS-CFP2 in SW620 (left) and TZM-bl (right) -derived cells and the same cell lines treated with 1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF. Tests were performed in triplicate. **p<0.01; ***p<0.001; ****p<0.0001; t-test.

Article Snippet: Cells with PS-CFP2 fluorescence were sorted from the TZM-blB8-mCherryB10-CFP cells following 72 h stimulation with 1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF (both from R&D systems, Minneapolis, MN, USA).

Techniques: Derivative Assay, Flow Cytometry, Control, Recombinant, Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: Next-generation cell lines for profiling different proteasome forms and their implications in cancer

doi: 10.3389/fimmu.2025.1672000

Figure Lengend Snippet: Validation of SW620B8-mCherryB5-GFP and TZM-blB8-mCherryB10-CFP cell lines. (A) Relative expression levels of the PSMB5, PSMB8 genes in SW620 and the PSMB5-EGFP , PSMB8-mCherry chimeras in SW620B8-mCherryB5-GFP cells, respectively. Gene expression was assessed in control and cytokine-treated (IFN-γ (1000 U/mL), TNF (500 U/mL)) cells. (B) Relative expression levels of the PSMB10 , PSMB8 , genes in TZM-bl cells and the PSMB10-PS-CFP2 , PSMB8-mCherry genes in TZM-blB8-mCherryB10-CFP cells. mRNA samples were obtained from control and IFN-γ/TNF-treated cells. Primers to endogenous PSMB5 , PSMB8 and PSMB10 genes were used for PCR with mRNA from initial cell lines, while primers targeting chimeras were utilized to study gene expression in modified cells ( Supplementary Table 1 ). *p < 0.05; ****p < 0.0001; t-test. (C) (Left) Western blotting of lysates obtained from SW620, SW620B8-mCherry and SW620B8-mCherryB5-GFP, and the same cells incubated with 1000 U/mL IFN-γ and 500 U/mL TNF for 72 h. (Right) Western blotting of lysates obtained from unstimulated TZM-bl, TZM-blB8-mCherry and TZM-blB8-mCherryB10-CFP, and the same cells treated with 1000 U/mL IFN-γ and 500 U/mL TNF for 72 h. To ensure an equal protein load, the membranes were stripped and then incubated with anti-β-actin antibodies. For PS-CFP detection, anti-GFP antibodies (Cell Signaling, Boston, MA, USA) were used. (D) (Left) Western blotting of proteasomes immunoprecipitated via the α4 subunit from homogenates of SW620 and derived cell lines using a Proteasome purification kit (Enzo, Farmingdale, NY, USA). (Right) Western blotting of proteasomes immunoprecipitated via α4 subunits from homogenates of TZM-bl and derived cell lines treated with IFN-γ/TNF for 72 h. The Proteasome purification kit (Enzo) was used to isolate proteasomes. The PS-CFP2 was revealed using anti-GFP (Cell Signaling) antibodies. The unbound fractions are marked as Unb. (E) Visualization of catalytically active proteasome subunits in cellular lysates. Homogenates of unstimulated and cytokine-treated (1000 U/mL IFN-γ and 500 U/mL TNF for 72 h) control and modified cells were incubated for 2 h at 37 °C with the Me 4 BodipyFL-Ahx 3 Leu 3 VS proteasome activity-based probe. The catalytic proteasome subunits were identified in 12% Tris-Glycine polyacrylamide gel following the excitation at a wavelength of 480 nm and an emission wavelength of 530 nm (left panel). The right panel shows the same gel stained with ROTI ® Blue quick protein stain (Carl Roth, Karlsruhe, Germany). (F) (Left) Western blotting of proteasomes immunoprecipitated via the α4 subunit from homogenates of SW620 and derived cell lines using a Proteasome purification kit (Enzo). Anti-mouse heavy chain HRP conjugates were used as secondary antibodies (ABclonal, Woburn, MA, USA). (Right) Western blotting of proteasomes immunoprecipitated from homogenates of IFN-γ/TNF-treated TZM-blB8-mCherryB10-CFP cells using antibodies to mCherry (Abcam, Cambridge, UK). Protein A agarose beads were used for the immunoprecipitation. (G) (Left) Western blotting of cellular homogenates and proteasomes immunoprecipitated using Protein A resin and anti-mCherry antibodies (Abcam) from homogenates of TZM-bl and derived cell lines, treated with IFN-γ/TNF for 72 h. Anti-rabbit light chain HRP conjugates (Cell Signaling) were used to detect primary antibody binding. (Right) Western blotting of TZM-bl-derived cell lines homogenates and proteasomes immunoprecipitated using Protein A resin and anti-mCherry antibodies (Abcam) from lysates of cytokine-treated TZM-blB8-mCherryB10-CFP cells. *Anti-rabbit heavy chain HRP conjugates (GeneTex, Irvine, CA, USA) were used as secondary antibodies. Specific bands are shown by red arrows and unspecific bands are shown by blue arrows.

Article Snippet: Cells with PS-CFP2 fluorescence were sorted from the TZM-blB8-mCherryB10-CFP cells following 72 h stimulation with 1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF (both from R&D systems, Minneapolis, MN, USA).

Techniques: Biomarker Discovery, Expressing, Gene Expression, Control, Modification, Western Blot, Incubation, Immunoprecipitation, Derivative Assay, Purification, Activity Assay, Staining, Binding Assay

Journal: Frontiers in Immunology

Article Title: Next-generation cell lines for profiling different proteasome forms and their implications in cancer

doi: 10.3389/fimmu.2025.1672000

Figure Lengend Snippet: Immune and intermediate proteasomes accumulate in the nuclei of TZM-blB8mCherryB10-CFP cells following stimulation with pro-inflammatory cytokines. (A) Confocal microscopy of unstimulated TZM-bl, TZM-blB8-mCherry, TZM-blB8-mCherryB10-CFP cells, and the same cells treated with pro-inflammatory cytokines. Twenty four hours after seeding, 1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF were added to the culture medium and the cells were incubated for an additional 72 h. After that, the cells were fixed in a 4% PFA solution. The photoconversion of PS-CFP2 was induced by intense 400 nm light irradiation. The fluorescence of the photoconverted protein was detected at 511 nm following excitation with a 488 nm light. Images are given in order from left to right: merged image, PS-CFP2 (blue channel), EGFP (green channel), mCherry (red channel). The fluorescence of PS-CFP2 can be seen in blue before photoconversion and in green after photoconversion. (B) (Left) The TZM-blB8-mCherryB10-CFP cells after photoconversion at a higher magnification. (Right) The Coloc 2 plugin FIJI (ImageJ) software 4 was used to perform pixel co-localization analysis, measuring the mean intensity value of pixels (0–255 for 8-bit images) within cell nuclei following photoconversion. The scale bar is 10 µm.

Article Snippet: Cells with PS-CFP2 fluorescence were sorted from the TZM-blB8-mCherryB10-CFP cells following 72 h stimulation with 1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF (both from R&D systems, Minneapolis, MN, USA).

Techniques: Confocal Microscopy, Recombinant, Incubation, Irradiation, Fluorescence, Software

Journal: Frontiers in Immunology

Article Title: Next-generation cell lines for profiling different proteasome forms and their implications in cancer

doi: 10.3389/fimmu.2025.1672000

Figure Lengend Snippet: Non-constitutive proteasomes form aggregates within the SW620B8-mCherryB5-GFP cells. (A) Confocal microscopy of unstimulated or IFN-γ/TNF-treated SW620 and SW620B8-mCherryB5-GFP cells. Pro-inflammatory cytokines (1000 U/mL of IFN-γ and 500 U/mL of TNF) were added to the culture media and the cells were left in the incubator for 72 h. After that, cells were fixed in a 4% PFA solution. To visualize cellular nuclei, cells were incubated with the NucBlue Fixed Cell ReadyProbe (Thermo Fisher Scientific, Waltham, MA, USA) (blue fluorescence). The images are given in order from left to right: mCherry (red channel), EGFP (green channel), merged image. Scale bar – 25 µm. (B) Magnification of unstimulated SW620B8-mCherryB5-GFP cells. Different combinations of fluorescence channels are shown. Non-constitutive proteasome aggregates are indicated by white arrows. The mCherry fluorescence is shown in red, the EGFP – in green. Merging of particular channels is indicated by “+”. Cellular nuclei are shown in blue. Scale bar – 10 µm. (C) Multiphoton microscopy of unstimulated or IFN-γ/TNF-treated live SW620 (left) and SW620B8-mCherryB5-GFP (right) cells. The nonlinear microscopy setup was optimized for the most distinct imaging of EGFP and mCherry, which was achieved by alternately two-photon excitation of the sample with pulses at wavelengths of 880 nm and 1045 nm, with emission detection in orthogonal spectral channels at 500–550 nm and 590–665 nm. The images are presented in order from left to right: mCherry (red channel), EGFP (green channel), and merged image. The SW620 and SW620B8-mCherryB5-GFP cells were cultured in DMEM lacking phenol red. Scale bar – 25 µm.

Article Snippet: Cells with PS-CFP2 fluorescence were sorted from the TZM-blB8-mCherryB10-CFP cells following 72 h stimulation with 1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF (both from R&D systems, Minneapolis, MN, USA).

Techniques: Confocal Microscopy, Incubation, Fluorescence, Microscopy, Imaging, Cell Culture