Fig. 4.

KLF9-dependent ROS regulate BRAF^V600E activation of ERK1/2 and proliferation in NHM. a NHMs infected with an empty vector (V) or BRAF^V600E-expressing vector (BRAF^V600E) were probed in immunoblotting with indicated antibodies (representative immuno-blots shown). Molecular weights are indicated on the right in kDa. b NHMs described in (a) were counted in triplicates on indicated days using trypan blue exclusion assay starting 48 h after infection (*p < 0.05; Student’s t-test). c Cells described in (a) were stained with H₂DCFDA (DCF) followed by FACS analysis to determine intracellular ROS. d NHMs were infected with control (Cl) or KLF9 (K9) shRNAs followed by transduction with empty vector (V) or BRAF^V600E-expressing vector (BRAF^V600E). Cells were probed in immunoblotting with indicated antibodies 48 h after second infection. Molecular weights are indicated on the right in kDa. e, f NHMs described in (d) were counted on indicated days in triplicates using trypan blue exclusion assay starting 48 h after infection (*p <0.05; Student’s t-test). g Cells described in (d) were stained with H₂DCFDA (DCF) followed by FACS analysis to determine intracellular ROS. h NHMs were infected with control (Cl) or KLF9 (K9) shRNAs followed by transduction with empty vector (V) or BRAF^V600E-expres-sing vector (BRAF^V600E). Cells were probed in immunoblotting with indicated antibodies 48 h after infection. pERK and ERK signals were quantified using ImageQuant software. Molecular weights are indicated on the right in kDa. pERK/ERK signal ratio was identified and normalized by that in “Vec/Cl” lane. i NHMs infected with the empty vector (Vec) or BRAF^V600E-expressing vector (BRAF^V600E) were treated with 0.5 mM NAC for 48 h and probed in immunoblotting with antibodies designated on the left. Molecular weights are indicated on the right in kDa. pERK and ERK signals were quantified using ImageQuant software. pERK/ERK signal ratio was calculated and normalized to that of vector control (“Vec” lane). j NHMs infected with empty vector (Vec) or KLF9-expressing vector (KLF9) were treated or not with 0.5 mM NAC for 48 h and probed in immunoblotting with indicated antibodies. Molecular weights are indicated on the right in kDa. k Cells described in (j) were stained with H₂DCFDA (DCF) followed by FACS analysis to determine intracellular ROS. 1 Cells described in (j) were cultured in media containing 0.5 mM NAC and were counted in triplicates on indicated days using to trypan blue exclusion assay starting 48 h after infection (*p <0.05; Student’s t-test). m, n ChIP assay. QPCR signals in reactions with DNA that was precipitated with NRF2, KLF9, or control IgG antibodies from NHMs expressing Vector or BRAF^V600E. QPCR was performed with primers encompassing NRF2 binding site in KLF9 promoter (ARE1) (ARE2) or KLF9-binding site in TXNRD2 promoter. All QPCR signals were normalized by those obtained from IgG-precipitated DNA in Vector cells. Representative experiment shown (*n = 3, technical replicas, p <0.05; Student’s t-test). o NHMs expressing Vector or BRAF^V600E were probed in QRT-PCR with primers corresponding to indicated genes. QRT-PCR signals were normalized by β-actin signal and by the corresponding signals in Vector cells. Representative experiment shown, (*n = 3, technical replicas, p <0.05; Student’s t-test). Each experiment was performed at least two times with consistent results