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. 2021 Jul 5;74(4):2007–2020. doi: 10.1002/hep.31888

FIG. 2.

FIG. 2

Effects of ERK5 silencing on iCCA cell proliferation. (A) ERK5 knockdown (shERK5) or nontargeting shRNA‐transfected (shNT) HuCCT‐1 cells were starved for 24 hours and incubated in presence of FBS or 100 ng/mL EGF, as indicated. Cell growth was evaluated after 48 hours by cell count and is represented as fold increase over 1% FBS/shNT. *P ≤ 0.05 versus shNT/1% FBS, **P ≤ 0.05 versus paired shNT (n = 4). (B) ERK5 knockdown (shERK5) or nontargeting shRNA‐transfected (shNT) CCLP‐1 cells were starved for 24 hours and incubated in absence or presence of 10% FBS, as indicated. Cell growth was evaluated after 48 hours by cell count and is represented as fold increase over control shNT. *P ≤ 0.05 versus shNT/control, **P ≤ 0.05 versus paired shNT (n = 4). (C) shNT or shERK5‐transfected HuCCT‐1 cells were starved for 24 hours and then incubated in presence or absence of 10% FBS or 100 ng/mL EGF for 24 hours. BrdU incorporation is represented as fold increase over control condition. *P ≤ 0.05 versus shNT/control, **P ≤ 0.05 versus paired shNT control (n = 4). (D) shNT or shERK5‐transfected CCLP‐1 cells were starved for 24 hours and then incubated in presence of 10% FBS. MTT analysis was conducted after 48 hours. *P ≤ 0.05 versus shNT/control, **P ≤ 0.05 versus paired shNT (n = 4). (E) ERK5 knockdown (shERK5) or nontargeting shRNA‐transfected (shNT) CCLP‐1 cells were starved for 24 hours and then incubated in absence or presence of 10% FBS or 100 ng/mL EGF. MTT analysis was conducted after 48 hours. *P ≤ 0.05 versus shNT/control, **P ≤ 0.05 versus paired shNT (n = 4). (F) HuCCT‐1 and CCLP‐1 cells were silenced for ERK5 using specific shRNA (shERK5) or with shNT. Immunoblotting with antibodies recognizing ERK5, p27, or proliferating cell nuclear antigen (PCNA) was performed. Equal loading of the gel was assessed by vinculin or glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) expression. Densitometries of p27 and PCNA expression were measured (n = 4) and are shown in the graphs. *P ≤ 0.05 versus shNT/control. Abbreviation: NT, nontargeting.