Journal: Cancers

Article Title: On the Therapeutic Potential of ERK4 in Triple-Negative Breast Cancer

doi: 10.3390/cancers15010025

Figure Lengend Snippet: Expression of ERK4 mRNA and protein in TNBC cell lines. ( A ) Expression of MAPK4 and MAPK6 mRNA was measured by real-time qPCR in the human TNBC cell lines Hs578T, MDA-MB231 and HCC1937, the breast epithelial cell line MCF10A, and control H1299, HeLa and HEK 293T cells. Results are expressed as Ct. Data are means ± SD ( n = 3). ( B ) Expression of MAPK4 and MAPK6 mRNA in Hs578T and MDA-MB-231 measured by RNA-seq. Results are expressed as TPM and correspond to the mean ± SD of triplicate samples. ( C) Expression of ERK4 and ERK3 proteins were analyzed by Western blotting using the indicated antibodies. ERK4 was depleted from HeLa cells by siRNA. ( D ) Hs578T were transfected with HA-ERK4 and analyzed by Western blotting with the indicated antibodies. ( E ) Expression of the ERK3 protein was analyzed by Western blotting. ( F ) Expression of MAPK4 and MAPK6 mRNA in human TNBC cell lines and H1299 NSCLC cell line extracted from the CCLE database. ( G ) MAPK4 gene dependency of breast cancer cell lines. The gene dependency score of MAPK4 was estimated from a CRISPR/Cas9 loss-of-function screen of human cancer cell lines and extracted from the DepMap portal ( https://depmap.org/portal/gene/MAPK4?tab=dependency ; accessed on 19 October 2022). A cell line is considered dependent if the dependency score is <−0.5. A score of 0 indicates a gene that is not essential. The median scores of essential genes is −1.0.

Article Snippet: Next day, the cells were transfected with HiPerfect and 3 μL of 10 μM ERK4 siRNA from Santa Cruz Biotechnology (sc-62280) or control siRNA from Qiagen (1027280).

Techniques: Expressing, Control, RNA Sequencing, Western Blot, Transfection, CRISPR

Journal: Cancers

Article Title: On the Therapeutic Potential of ERK4 in Triple-Negative Breast Cancer

doi: 10.3390/cancers15010025

Figure Lengend Snippet: ERK4 catalytic activity has no impact on the phosphorylation of AKT on Thr308 and Ser473. ( A ) Hs578T TNBC cells were transfected with a GFP-expressing plasmid to show the high yield of transfection of the cells. ( B ) Hs578T cells were transfected with HA-ERK4 or catalytically-inactive HA-ERK4 KK49/50AA with or without Flag-MK5. Phosphorylation of endogenous AKT on Thr308 and Ser473 or of ectopically expressed Flag-MK5 was analyzed by immunoblotting with phospho-specific antibodies. Expression of HA-ERK4 was measured with anti-HA antibody. ( C ) HEK 293T cells were transfected with GFP-ERK4 or catalytically-inactive GFP-ERK4 KK49/50AA with or without GFP-MK5. Phosphorylation of endogenous AKT on Thr308 and Ser473 or of ectopically expressed GFP-MK5 was analyzed by immunoblotting with phospho-specific antibodies. Expression of GFP constructs was controlled with anti-GFP antibody.

Article Snippet: Next day, the cells were transfected with HiPerfect and 3 μL of 10 μM ERK4 siRNA from Santa Cruz Biotechnology (sc-62280) or control siRNA from Qiagen (1027280).

Techniques: Activity Assay, Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Western Blot, Construct