Fig. 4: The MEK5/ERK5 pathway controls the proliferation of human sarcoma cell lines.

a ERK5 expression was evaluated by immunoprecipitation of 1 mg of protein from sarcoma cell extracts with the anti-ERK5 Pro1 antibody followed by Western blotting with the anti-ERK5 C-terminal antibody. Seventy micrograms of protein extract was used to detect MEK5. Calnexin was used as a loading control. b ShRNA sequences sh66 and sh68 were used to decrease MEK5 protein levels in the different sarcoma cell lines, and MEK5 protein expression was compared with that in cells treated with shC (noncoding sequence) by Western blotting using 70 µg of cell extracts. The antiproliferative effect of the knockdown was analyzed by a cell counting assay when the control cells reached 85-90% confluence, and the proliferation rate is presented as a percentage of that in shC cells. Data are presented as the mean ± SD of two independent experiments with four biological replicates each. p-values: ***p ≤ 0.001. c Knockdown of ERK5 expression was carried out with shRNA sequences sh62 and sh75. ERK5 protein levels were analyzed by immunoprecipitation of 1 mg of protein with the anti-ERK5 Pro1 antibody followed by Western blotting with the anti-ERK5 C-terminal antibody. Calnexin was used as a loading control. The effect of ERK5 knockdown on cell proliferation was quantified by cell counting as described above, and the proliferation rate is presented as a percentage of that in shC cells. Data are presented as the mean ± SD of two independent experiments with four biological replicates each. p-values: **p ≤ 0.01; ***p ≤ 0.001. d SKUT1-ERK5 knockout clones (#2, #5, #30 and #42) were obtained by using CRISPR/Cas9 gene editing. The lack of ERK5 expression was determined by immunoprecipitation of 1 mg of protein extract followed by Western blotting with the anti-ERK5 antibody. Calnexin was used as a loading control. ERK5 KO clones and Sc cells were cultured for 5 days in 6-well plates, and the proliferation rate was measured by cell counting and is presented as a percentage of that in Sc cells. Data are presented as the mean ± SD of two independent experiments with four biological replicates each. p-values: ***p ≤ 0.001. e SKLMS1 ERK5 knockout clones (#3, #11) were also obtained. The lack of ERK5 expression was analyzed as described above. Cell counting was performed after 4 days of culture to study the effect on cell proliferation. Data are presented as the mean ± SD of two independent experiments with four biological replicates each. p-values: ***p ≤ 0.001.