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. 2021 Dec 3;20(12):2317–2328. doi: 10.1158/1535-7163.MCT-21-0367

Figure 3.

Figure 3. JNJ-64619178 promotes cellular PRMT5 thermal shift stabilization and leads to prolonged pathway inhibition after compound washout. A, NCI-H1048 cells were treated with JNJ-64619178 (0.1 μmol/L) or Cpd-2 (10 μmol/L) for either 1 hour (1-hour treatment), 1 hour followed by compound washout and further incubation in compound-free complete media for 72 hours (after washout), or continuously for 72 hours (continuous treatment). Thermal shift (60°C for 3 minutes) was applied, and soluble proteins (supernatant) were separated by WES (ProteinSimple). PRMT5 levels were quantitated, normalized to β-actin and plotted. Dashed red rectangle highlights the washout treatment. Error bars represent mean ± standard deviation (SD). B, PRMT5 downstream signalling in NCI-H1048 cells after compound treatment assessed by immunoblotting against SmD1/3-Me2, SmD3, PRMT5, and MEP50. Cells were either treated with JNJ-64619178 (0.03 and 0.3 μmol/L) or Cpd-2 (0.3 and 3.0 μmol/L) or DMSO (No treatment) as indicated. Dashed red rectangle highlights the washout treatment. C, Inhibition of cell proliferation under intermittent (wash out; y-axis) and continuous (no wash out; x-axis) treatment with JNJ-64619178 (10 pmol/L–1 μmol/L dose response) was assessed over 10 days and AUC (% inhibition/nmol/L) was plotted for 94 cancer cell lines from multiple cancer types (each cancer type represented by different color dots). For washout condition, exponentially growing cells were treated for 3 days with JNJ-64619178, followed by extensive compound washout. Cell growth inhibition was determined after 7 additional days after washout. For the 10-day continuous compound treatment condition (no wash out), cells were continuously treated with JNJ-64619178 at the same concentrations as described above. Linear regression (slope = 0.873) was determined (Pearson's correlation = 0.993).

JNJ-64619178 promotes cellular PRMT5 thermal shift stabilization and leads to prolonged pathway inhibition after compound washout. A, NCI-H1048 cells were treated with JNJ-64619178 (0.1 μmol/L) or Cpd-2 (10 μmol/L) for either 1 hour (1-hour treatment), 1 hour followed by compound washout and further incubation in compound-free complete media for 72 hours (after washout), or continuously for 72 hours (continuous treatment). Thermal shift (60°C for 3 minutes) was applied, and soluble proteins (supernatant) were separated by WES (ProteinSimple). PRMT5 levels were quantitated, normalized to β-actin and plotted. Dashed red rectangle highlights the washout treatment. Error bars represent mean ± standard deviation (SD). B, PRMT5 downstream signalling in NCI-H1048 cells after compound treatment assessed by immunoblotting against SmD1/3-Me2, SmD3, PRMT5, and MEP50. Cells were either treated with JNJ-64619178 (0.03 and 0.3 μmol/L) or Cpd-2 (0.3 and 3.0 μmol/L) or DMSO (No treatment) as indicated. Dashed red rectangle highlights the washout treatment. C, Inhibition of cell proliferation under intermittent (wash out; y-axis) and continuous (no wash out; x-axis) treatment with JNJ-64619178 (10 pmol/L–1 μmol/L dose response) was assessed over 10 days and AUC (% inhibition/nmol/L) was plotted for 94 cancer cell lines from multiple cancer types (each cancer type represented by different color dots). For washout condition, exponentially growing cells were treated for 3 days with JNJ-64619178, followed by extensive compound washout. Cell growth inhibition was determined after 7 additional days after washout. For the 10-day continuous compound treatment condition (no wash out), cells were continuously treated with JNJ-64619178 at the same concentrations as described above. Linear regression (slope = 0.873) was determined (Pearson's correlation = 0.993).