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. 2023 Mar 14;11(3):e006325. doi: 10.1136/jitc-2022-006325

Figure 3.

Figure 3

NSD2 enhances RARα liquid–liquid phase separation in myeloma cells. (A) Prediction of IDRs (green frame) of RARα protein using a PONDR algorithm. (B) Visualization of turbidity associated with droplet formation. Tubes containing GFP (left), GFP–RARα–IDR1, and GFP–RARα–IDR2 (middle and right) in the presence of PEG8000. (C) Turbidity (OD600) of GFP–RARα–IDR1 and GFP–RARα–IDR2 in 20% PEG. (D) Representative images (n=3 biologically independent experiments) of droplet formation of GFP–RARα–IDR1 and IDR2 in MM cells at different salt concentrations. GFP–RARα–IDR1 and IDR2 were added to droplet formation buffer to achieve 5 µM protein concentration with a final NaCl concentration as indicated. (E) Turbidity of GFP–RARα–IDR1 and GFP–RARα–IDR2 in different salt concentrations as indicated. (F) Phase separation formation of FL GFP–RARα in MM cells before and after treatment with 4% 1,6-hexanediol at different times. Scale bar, 5 µm. (G) FRAP of a RARα–GFP focus (red framed) by 488 nm laser for 10″ bleaching and 90″ recovery in KMS11 cells. Scale bar, 5 µm. (H) Kinetic recovery times of bleached GFP-fusion RARα droplet foci in MM cells by 488 nm laser. Two-sided p values of the comparisons between the final extents of recovery after photobleaching were performed using one-way analysis of variance; mean±SD.d. (I) Visualization of turbidity associated with droplet formation. Tubes containing GFP (left) and GFP–RARα–ΔIDRs (right) in the presence of PEG8000 (J) KMS11 cells were infected with lentivirus carrying GFP–RARα–ΔIDRs-over expression plasmid for 72 hours and the GFP-fusion protein were detected using IF. (K) Representative images for endogenous foci of RARα in vector control and NSD2 KD KMS11 and LP-1 cells. Scale bar, 5 µm. (L, M) Representative images of droplet formation of GFP-fusion RARα-IDR1 and IDR2 with 5 µM protein concentration in the presence or absence of NSD2 at different NaCl concentrations as indicated. (N) Kinetic recovery times of bleached GFP-fusion RARα droplet foci in HEK293T cotransfected with NSD2 expressing vector (NSD2-OE) or sgRNA (NSD2 KD) by 488 nm laser (n=3 biologically independent experiments). Two-sided p values of the comparisons between the final extents of recovery after photobleaching were performed using one-way analysis of variance; mean±SD. FL, full length; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; IDR, intrinsically disordered region; IF, immunofluorescence; KD, knocked-down; MM, multiple myeloma.