Journal: Cancers

Article Title: Dual Targeting of EZH2 Degradation and EGFR/HER2 Inhibition for Enhanced Efficacy against Burkitt’s Lymphoma

doi: 10.3390/cancers15184472

Figure Lengend Snippet: Cell viability after treatment with MS1943 and lapatinib was measured using the CCK-8 assay. ( A ) The Ramos cell line was exposed to 2.5 μM, 5 μM, and 10 μM of MS1943 and observed for 24, 48, and 72 h. ( B ) Meaningful results were shown in 72 h with 2.5 μM, 5 μM, and 10 μM of treatment. ( C ) In the Daudi cell line, MS1943 was treated with different concentrations as described above for 24, 48, and 72 h. ( D ) Treatment of MS1943 in the Daudi cell line exhibited dose-dependent inhibition for 72 h. ( E ) Ramos cells were exposed to 2.5 μM, 5 μM, and 10 μM of lapatinib and observed for 24, 48, and 72 h. ( F ) Ramos cell viability indicated dose-dependent inhibition in 72 h. ( G ) In the Daudi cell line, lapatinib was treated with different concentrations as described above for 24, 48, and 72 h. ( H ) Treatment of lapatinib in the Daudi cell line led to dose-dependent inhibition for 72 h. *, p < 0.05; **, p < 0.01; ***, p < 0.001 as determined by two-tailed, unpaired t -tests. Error bars are shown as mean ± SD.

Article Snippet: Lapatinib (HER2/neu-EGFR inhibitor) and MS1943 (EZH2 degrader) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: CCK-8 Assay, Inhibition, Two Tailed Test

Journal: Cancers

Article Title: Dual Targeting of EZH2 Degradation and EGFR/HER2 Inhibition for Enhanced Efficacy against Burkitt’s Lymphoma

doi: 10.3390/cancers15184472

Figure Lengend Snippet: Drug combination therapy for 72 h showed synergistic effects in the CCK-8 assay. ( A ) The Ramos cell line was exposed to 5 μM of MS1943 and lapatinib each or in combination. Combination treatment exhibited a significant reduction compared with the DMSO control and single-treated drug. ( B ) Daudi cell viability was assayed using CCK-8 analysis and the represented co-treatment demonstrated promising effects against the control and single therapy. *, p < 0.05; ***, p < 0.001 as determined by two-tailed, unpaired t -tests. Error bars are shown as mean ± SD.

Article Snippet: Lapatinib (HER2/neu-EGFR inhibitor) and MS1943 (EZH2 degrader) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: CCK-8 Assay, Control, Two Tailed Test

Journal: Cancers

Article Title: Dual Targeting of EZH2 Degradation and EGFR/HER2 Inhibition for Enhanced Efficacy against Burkitt’s Lymphoma

doi: 10.3390/cancers15184472

Figure Lengend Snippet: Cell cycle analysis revealed apoptosis by arresting S phases. ( A ) Ramos cells were treated with control, MS1943, and lapatinib for each or in combination for 72 h and detected by flow cytometry. ( B ) The relative ratio of S phases expanded in both MS1943 and lapatinib treatment. S phase-arrest indicates apoptosis. ( C ) The synergistic effects of MS1943 and lapatinib induced cell cycle arrest through Cyclin D1 upregulation in Ramos cells. ( D ) Daudi cells were exposed in the same fashion as the Ramos cells. ( E ) The relative ratio of S phases largely increased compared to the control and single-treated cells, representing cell death by apoptosis. ( F ) The synergistic effects of MS1943 and lapatinib induces cell cycle arrest through Cyclin D1 upregulation in lymphoma. The experiment was repeated in duplicate and merged data from all the experiments are shown. **, p < 0.01; as determined by two-tailed, unpaired t -tests. Error bars are shown as mean ± SD.

Article Snippet: Lapatinib (HER2/neu-EGFR inhibitor) and MS1943 (EZH2 degrader) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Cell Cycle Assay, Control, Flow Cytometry, Two Tailed Test

Journal: Cancers

Article Title: Dual Targeting of EZH2 Degradation and EGFR/HER2 Inhibition for Enhanced Efficacy against Burkitt’s Lymphoma

doi: 10.3390/cancers15184472

Figure Lengend Snippet: Apoptotic effects of combination treatment on Ramos and Daudi cell were examined by Annexin V staining. Each section indicates Necrosis cells (Q1), Late Apoptotic cells (Q2), Early Apoptotic cells (Q3) and Live cells (Q4). ( A ) Ramos cells were treated with 5 µM of control, lapatinib, MS1943, and a combination thereof. Apoptosis was evaluated after 72 h of treatment. ( B , C ) The relative percentage of Early Apoptotic cells (Q3) and Late Apoptotic cells (Q2) were meaningfully increased compared with the control. ( D ) Daudi cells were treated in the same way as Ramos cells. ( E , F ) The relative percentages of Early Apoptotic cells (Q3) and Late Apoptotic cells (Q2) were significantly increased against the control. These showed that dual-targeting therapeutic combinations induced apoptosis effectively. The experiment was repeated in duplicate and merged data from all the experiments are shown. *, p < 0.05; **, p < 0.01; as determined by two-tailed, unpaired t -tests. Error bars are shown as mean ± SD.

Article Snippet: Lapatinib (HER2/neu-EGFR inhibitor) and MS1943 (EZH2 degrader) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Staining, Control, Two Tailed Test

Journal: Cancers

Article Title: Dual Targeting of EZH2 Degradation and EGFR/HER2 Inhibition for Enhanced Efficacy against Burkitt’s Lymphoma

doi: 10.3390/cancers15184472

Figure Lengend Snippet: Drug combination induced upregulation of apoptosis-associated proteins. ( A ) The Ramos samples were exposed to 5 μM MS1943 and lapatinib each or in combination for 72 h. ( B – D ). Western blot results were analyzed relatively by using GAPDH. All the proteins associated with apoptosis were highly increased in both MS1943- and lapatinib-treated cells. ( E ) Combination treatment in the Daudi cells exhibited distinctive outcomes with increased relative mRNA expression of p53. ( F ) Daudi cells were exposed to the same 5 μM as mentioned above for 72 h. ( G – I ) Western blot results were analyzed relatively and indicated upregulation in the combination therapeutic agents. This demonstrates that the drug combination induced apoptosis effectively in protein levels. ( J ) Combination treatment in the Daudi cells showed distinctive outcomes with increased relative mRNA expression of p53. Western blot total gel can be found in . *, p < 0.05; **, p < 0.01; ***, p < 0.001 as determined by two-tailed, unpaired t -tests. Error bars are shown as mean ± SD.

Article Snippet: Lapatinib (HER2/neu-EGFR inhibitor) and MS1943 (EZH2 degrader) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: Cell Genomics

Article Title: Single-cell genome-wide association reveals that a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus

doi: 10.1016/j.xgen.2022.100207

Figure Lengend Snippet: A nonsynonymous variant in ERAP1 regulates IAV burden in cells (A) Stratified QQ plot for association with mean IAV reads restricted to RASQUAL 2-step FDR eQTLs (FDR < 0.05; MAF > 0.1). Plotting eQTLs reveals an SNP associated with expression of TNFSF12 that has a lower p value than expected by chance. (B) Stratified QQ plot for association with mean IAV reads restricted to nonsynonymous SNPs. Plotting nonsynonymous variants (MAF >0.1) reveals an SNP in ERAP1 that has a p value lower than expected by chance. (C) Genotype median plot for mean IAV reads as a function of rs27895 genotype. P value from EMMAX and corrected for genomic inflation factor. (D) RNAi against TNFSF12 in either NA19399 (TT for rs12103519) or NA19328 (CC for rs12103519) demonstrates reducing TNFSF12 expression (mean 87% knockdown ±4% [SEM] for NA19328 and mean 73% knockdown ±6% [SEM] for NA19399) does not significantly affect the percentage of IAV-infected cells at 24 h in either LCL. Replicates from 3 separate experiments were normalized to correct for between-experiment variation. p values are from unpaired t tests. (E) RNAi against ERAP1 in either NA19020 (TT for rs27895) or NA19399 (CC for rs27895) demonstrate that reducing ERAP1 expression (mean 80% knockdown ±14% [SEM] for NA19020 and mean 70% knockdown ±8% [SEM] for NA19399) decreases the percentage of IAV-infected cells at 24 h in both LCLs. Replicates from 4 separate experiments were normalized to correct for between-experiment variation. One experiment involving NA19020 was excluded because no knockdown was detected. p values are from unpaired t tests. (F) Specific ERAP1 inhibitor, ERAP1-IN-1 (CAS: 865273-97-8, MedChemExpress) demonstrates dose-dependent reduction in the percentage of IAV-infected cells at 24 h in A549s. Replicates from 3 separate experiments were normalized to correct for between-experiment variation. p values are from ordinary one-way ANOVA with Dunnett’s multiple comparisons test using DMSO (vehicle) control. (G) Interaction between ERAP1 residue 346 and an ERAP1 peptide inhibitor. Ribbon diagram of the crystal structure of ERAP1 (light gray, cartoon) bound to a 10-mer peptide inhibitor (purple) (PDB: 6RQX ). The carbon atoms of ERAP1 residue 346 and the sixth position of the peptide are shown as orange-, yellow- and purple-colored spheres, respectively. (H) Wild-type ERAP1 G346 and F6 of the 10-mer peptide inhibitor have multiple favorable side chain-backbone van der Waals contacts. (I) However, steric clash between the ERAP1 side chain and the 10-mer peptide inhibitor occurs when G346 is mutated to an aspartate (carbon atoms colored green) using in silico mutagenesis. (J) Rare rotamers of ERAP1 G346D do not clash with a peptide in which F6 is replace by a glycine but provide no favorable contacts. (K) Even the addition of only a β carbon at position 6 of the 10-mer peptide inhibitor, i.e., an alanine residue, clashes with ERAP1 (G346D). (L) Overexpression of alternative alleles of ERAP1 in A549 cells demonstrates that the T allele of rs27895 (aspartate) increases the percentage of IAV-infected cells at 24 h. 8 biological replicates from 3 separate experiments were normalized to correct for between-experiment variation. p values are from ordinary one-way ANOVA with Tukey’s multiple comparisons test. Overexpression of alternative alleles of ERAP1 was confirmed by western blot. (M) Model of how rs27895 affects ERAP1 ’s transcript and protein and the resulting effect on viral burden. The reference allele of rs27895 encodes cytosine on the genome reference strand and guanine on the transcribed strand. This reference allele encodes glycine at position 346 of ERAP1 and is associated with reduced viral burden in cells. The alternate allele of rs27895 encodes thymine on the genome reference strand and adenine on the transcribed strand. This alternate allele encodes aspartate at position 346 of ERAP1 and is associated with increased viral burden in cells

Article Snippet: One experiment involving NA19020 was excluded because no knockdown was detected. p values are from unpaired t tests. (F) Specific ERAP1 inhibitor, ERAP1-IN-1 (CAS: 865273-97-8, MedChemExpress) demonstrates dose-dependent reduction in the percentage of IAV-infected cells at 24 h in A549s.

Techniques: Variant Assay, Expressing, Knockdown, Infection, Control, Residue, In Silico, Mutagenesis, Over Expression, Western Blot

Journal: Cell Genomics

Article Title: Single-cell genome-wide association reveals that a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus

doi: 10.1016/j.xgen.2022.100207

Figure Lengend Snippet: A nonsynonymous variant in ERAP1 regulates IAV burden and symptomology in human challenge (A) Flow chart of Prometheus study. (B) rs27895 T allele is associated with IAV burden at day 4 of the Prometheus study. Viral burden was measured by nasal lavage by qPCR using pan IAV M gene primers (see ). p values from EMMAX at each time point for rs27895 are listed below the plot. Mean and SD are plotted with lines connecting means. (C) rs27895 T allele is associated with greater symptomology at days 3–7. Jackson symptomology score was assessed daily. p values from EMMAX at each time point for rs27895 are listed below the plot. Mean and SD are plotted with lines connecting means

Article Snippet: One experiment involving NA19020 was excluded because no knockdown was detected. p values are from unpaired t tests. (F) Specific ERAP1 inhibitor, ERAP1-IN-1 (CAS: 865273-97-8, MedChemExpress) demonstrates dose-dependent reduction in the percentage of IAV-infected cells at 24 h in A549s.

Techniques: Variant Assay

Journal: Cell Genomics

Article Title: Single-cell genome-wide association reveals that a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus

doi: 10.1016/j.xgen.2022.100207

Figure Lengend Snippet:

Article Snippet: One experiment involving NA19020 was excluded because no knockdown was detected. p values are from unpaired t tests. (F) Specific ERAP1 inhibitor, ERAP1-IN-1 (CAS: 865273-97-8, MedChemExpress) demonstrates dose-dependent reduction in the percentage of IAV-infected cells at 24 h in A549s.

Techniques: Virus, Recombinant, Over Expression, TaqMan Assay, Software