Cell Viability and Apoptosis Assay

Cell viability of HCT116 cells was analyzed by MTT (Sigma, St. Louis, MO) assay. 4000 cells were seeded 24 hrs before either knockdown or transfection of cells with vectors containing p68 and the mutants. Subsequently, 24 hrs after transfection,

the cells were induced for apoptosis by oxaliplatin treatment. The next day, reconstituted MTT-reagent was added in an amount equal to 10% of the volume of the cell culture medium and incubated at 37°C. MTT Solubilization solution used to dissolve the formazan crystals formed during incubation was subsequently added. Cell viability was determined spectrophotometrically by measuring absorbance at A570. Cells plated

on 6-well plates were treated with 10 μM oxaliplatin after p68 knockdown and transfection and caspase-3/CPP32 colorimetric assay kit (Biovision Research Products, Milpitas, CA) was used for measuring caspase-3 cleavage. Briefly, after apoptosis induction, the cells were lyzed in Cell Lysis Buffer for 10 mins, centrifuged and subjected to cleavage assay. The reaction consisted of cell lysate diluted in Cell Lysis Buffer, 2X Reaction buffer, and 4 mM DEVD-pNA substrate. The samples were read at A405 using a microtiter plate reader. FITC-Annexin V Apoptosis Detection Kit

(BDBiosciences, San Jose, CA) was also used to measure apoptosis induced by oxaliplatin.

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0 20 40 80 Oxa concentration (µM) % ap o p to tic c e ll s IB: p68 IB: 14B3 Oxa (μM) 0 20 80 160 IP : p 68 Ad h e r e n t F lo at in g IB: 14B3 IB: 14B3 IB: p68 IB: p68 IP : p 68 Time (hrs) 0 1 2 4 6 24 IB: 14B3 IB: p68 IP : p 68 Oxa (μM) 0 20 80 160 IP : p 68 D B A C 0 10 20 30 40 50 60 70 1 2 3 4 % a po pt ot ic c e ll s Series1

Figure 2.1. Threonine phosphorylation of p68 in HCT116 cells following

oxaliplatin (Oxa) treatment.

(A) Apoptosis of HCT116 cells induced by different concentrations of oxaliplatin was measured and presented as percent of apoptotic cells after 24 hours treatment. Error bars represented standard deviations of four independent experiments.

(B), (C), and (D) Threonine phosphorylations of p68 measured by immunoblotting using anti-phospho-threonine antibody (14B3) (1:1000 dilution) in HCT116 cells that were treated with different oxaliplatin concentration (B) and (C), or 20 μM of oxaliplatin for

different times (D) were analyzed by immunoblotting the p68 that was immunoprecipitated (IP: p68) using antibody against anti-phospho-threonine (IB: 14B3). The immunoblot of p68 (IB: p68) in the immunoprecipitates represented the amount of p68 that was precipitated. In (C), the analyses were carried out with the cells that were already detached and the cells that were still adherent following the treatment for 24 hours.

Time (hrs) 0 1 2 4 6 24 IP : p 68 IB: 14B3 IB: p68 IB: p-p38 IB: p38 IB: p38 IB: p68 Oxa - + + Auto- radiography CBS _BSA FLAG-p38 BSA IB: p68 IB: FLAG IB: GAPDH A B

IP: p68 IP: IgG

p68 BSA+p 38 p68 p68+p 38 p38 C Vec D176A -F327L p38WT IB: 14B3 IP : p 68 D p68 p38

Figure 2.2 P68 Phosphorylation by p38 MAP-kinase

(A) Threonine phosphorylation of p68 in HCT116 cells following treatment of the cells with 20 μM of oxaliplatin for different times were analyzed by immunoblotting the p68 that was immunoprecipitated (IP: p68) using antibody against phospho-threonine (IB: 14B3) from the cell lysates. Phosphorylation of p38 MAP-kinase during the same treatment was analyzed by immunoblot of cell lysates using antibody against the phosphorylated p38. The immunoblot of p68 (IB: p68) in the immunoprecipitates represented the amount of p68 that was precipitated. The immunoblot of p38 in the cell lysate (IB: p38) indicated the cellular levels of p38 as a control.

(B) Coimmunoprecipitation of p38 and p68 in the cell extracts of HCT116 cells with/without oxaliplatin treatment (Oxa, +/-) was analyzed by p68 immunoblot with p68 antibody after immunoprecipitation (IP: p68) and by using antibody against p38 (IB: p38). The immunoblot of p68 (IB: p68) from the immunoprecipitates represented the amount of p68 that was precipitated. The IP: IgG was the immunoprecipitation using rabbit IgG, serving as a negative control IP.

(C) Phosphorylation of recombinant His-p68, or BSA as a control, by recombinant p38 was demonstrated by autoradiography. The amount of protein used in the phosphorylation reactions was shown by Coomassie Blue Staining (CBS).

(D) Phosphorylation of p68 by overexpression of Flag-tagged p38 MAPK, wild-type and constitutively active mutant D176A-F327L, in HCT116 cells was analyzed by immunoblotting the p68 that was immunoprecipitated (IP: p68) using antibody against phospho-threonine (IB: 14B3). The immunoblot of p68 (IB: p68) in immunoprecipitates represented the amount of p68 that was precipitated. The immunoblot of Flag-tag (IB: FLAG) indicated the overexpressed p38 levels. The immunoblot of GAPDH (IB: GAPDH) served as a loading control.

MEF2C Q S L A T P V V S MAPKAPK2 K V P Q T P L H T ATF-2 D Q T P T P T R F P68-T610 Y P M P T G Y S Q P68-T564 T G N P T G T Y Q P68-T446 Y T F F T P N N I P68-T224 I C I A T P G R L

WT T610A T564A T224A Autoradiography CBS Autoradiography CBS WT _{T564/446A T}446/224A IP : HA IB: 14B3 IB: p68 IP : HA IB: 14B3 IB: p68 Oxa - + - + - + - + Vec WT T564/446A T446/224A HA-p68 T564A

Vec WT T610A T556AT446AT224A

HA-p68 A C B His-p68 His-p68

Figure 2.3 Phosphorylation site(s) of p68 by p38 MAPK

(A) Prediction of potential p38 MAP-kinase-induced phosphorylation site(s) in the p68- reading frame and compared to consensus p38 MAPK phosphorylation sites of several authentic p38 MAP-Kinase substrates by a web-based phosphorylation-site prediction program NetPhos 2.0, (B) Phosphorylation of recombinant His-p68 and mutants with single site mutation (Upper) and double site mutations (Lower) by recombinant p38 was demonstrated by autoradiography. The amount of protein used in the phosphorylation reactions was shown by Coomassie Blue Staining (CBS). (C) Phosphorylation of overexpressed HA-p68s, wild type (WT) and mutants (Single site mutation, Upper, and Double site mutations, Lower), in HCT116 cells with/without oxaliplatin treatment (Oxa,

+/-) was analyzed by immunoblotting the p68 that was immunoprecipitated (IP: p68) using antibody against phospho-threonine (IB: 14B3). The immunoblot of p68 (IB: p68) from immunoprecipitates represented the amount of p68 that was precipitated.

Figure 2.4 Effects of p68 phosphorylation on cell apoptosis

(A) HCT116 cells were treated by non-targeting siRNA (NT) or siRNA targeting p68 (p68) to knockdown p68 for 24 hours. Wild type and the different mutants of p68 were overexpressed in the p68 knockdown cells. P68 (IB: p68) immunoblot represented the cellular levels of p68 (both endogenous and overexpressed). The immunoblot of GAPDH (IB: GAPDH) served as a loading control.

Cell apoptosis (B) and viability (C) of the cells in (A) that were treated or untreated with oxaliplatin were measured by a caspase-3 apoptosis kit (B) or MTT assay (C). The cell apoptosis and viability were presented as relative apoptosis and relative viability by defining the apoptosis and viability of the vector expressing cells without oxaliplatin treatment as 1. Error bars represented standard deviations of four independent experiments.