Journal: Molecular carcinogenesis

Article Title: FAM129B-dependent activation of NRF2 promotes an invasive phenotype in BRAF mutant melanoma cells

doi: 10.1002/mc.23295

Figure Lengend Snippet: For panels (A-D), A375 cells were transiently transfected with 10 nM of either control or FAM129B siRNA for 72 hr prior to experimentation. (A) Cells were scratched to create a wound; images shown are at time of initial scratch (0 h) and 24 hr later. (B) Percent wound closure was calculated every 6 hr up to 24 hr post scratch from the cells/images in (A) (n=3). (C) Cells were subjected to a chemotactic invasion assay; values shown are the relative number of cells that migrated into the lower well at 48 hr post seeding normalized to the initial number of cells in the upper well (n=3). (D) Immunoblot analysis of NRF2 and SOX9 protein levels. For panel (E-H) A375 cells were treated with 10 nM control or NRF2 siRNA for 72 hr, then 10 μM U0126 or 40 nM brusatol for 4 hours prior to collection. (E) Immunoblot analysis of NRF2, p-FAM129B, FAM129B, p-ERK, and ERK protein levels. (F) Cells were scratched and imaged every 6 hr for 24 hr. Images displayed represent initial scratch (0 h) and 24 hr later. (G) Wound percent closure was quantified from cells/images in (F) (n=3). (H) The cells were monitored for invasion via the chemotactic invasion assay. Invasion is represented by the number of cells that migrated to the lower well at 48 hr normalized to the initial number of cells in the upper well (n=3).

Article Snippet: Antibodies were purchased from Santa Cruz Biotechnology (NRF2, KEAP1, NQO1, BRAF, GAPDH, p-ERK, ERK, SOX9), Cell Signaling Technology (Tubulin, FAM129B), MyBioSource (p-FAM129B), BD Biosciences (β-catenin), New England Biolabs (CBD), and Trevigen (HA).

Techniques: Transfection, Control, Invasion Assay, Western Blot

Journal: Molecular carcinogenesis

Article Title: FAM129B-dependent activation of NRF2 promotes an invasive phenotype in BRAF mutant melanoma cells

doi: 10.1002/mc.23295

Figure Lengend Snippet: HEK293 cells were co-transfected with HA-FAM129B and KEAP1-CBD and protein-protein interaction was assessed via co-immunoprecipitation analysis. Cell lysates were incubated with either (A) HA-beads or (B) CBD beads and immunoprecipitated complexes were then immunoblotted using either anti-HA or anti-CBD antibodies. (C) HEK293 cells were cotransfected with either wild type or mutant HA-FAM129B in the presence or absence of KEAP1-CBD for 24 hr, and proteins in the KEAP1-containing complexes were pulled down with CBD beads, and then immunoblotted using either anti-HA or anti-CBD antibodies. (D) HEK293 cells were transfected with HA-FAM129B or HA-muFAM129B for 24 hr prior to collection; NRF2, HA-FAM129B, and NQO1 levels were determined via immunoblot analysis. (E) FAM129B or muFAM129B was co-transfected with NQO1-ARE-driven firefly luciferase and TK-driven-Renilla luciferase in HEK293 cells for 24 hr prior to measurement of dual luciferase activity. For both (D) and (E), cells were treated with 50 μM tBHQ for 24 hr as a positive control for NRF2 activation.

Article Snippet: Antibodies were purchased from Santa Cruz Biotechnology (NRF2, KEAP1, NQO1, BRAF, GAPDH, p-ERK, ERK, SOX9), Cell Signaling Technology (Tubulin, FAM129B), MyBioSource (p-FAM129B), BD Biosciences (β-catenin), New England Biolabs (CBD), and Trevigen (HA).

Techniques: Transfection, Immunoprecipitation, Incubation, Mutagenesis, Western Blot, Luciferase, Activity Assay, Positive Control, Activation Assay

Journal: Molecular carcinogenesis

Article Title: FAM129B-dependent activation of NRF2 promotes an invasive phenotype in BRAF mutant melanoma cells

doi: 10.1002/mc.23295

Figure Lengend Snippet: (A) A375 cells were transfected with 80 nM BRAF siRNA twice over 72 hours prior to immunoblot analysis of BRAF, p-FAM129B, FAM129B, and NRF2. (B) NRF2/GAPDH and BRAF/GAPDH protein levels were quantified from immunoblot analyses in (A) (n=3). (C) A375 cells were transiently transfected with either 80 nM control or BRAF siRNA twice over 72 hours prior to 4 hr treatment with 10 μM U0126 or 40 nM brusatol; cells were then analyzed using indirect immunofluorescence for FAM129B (left) and β-catenin (middle). (D) Indirect immunofluorescence analysis for FAM129B (left) and KEAP1 (middle) at 4 hr post 10 μM U0126 treatment; merged panel (right) indicates colocalization of FAM129B and KEAP1. (E) A375 cells were transfected with BRAF siRNA and then subjected to indirect immunofluorescence analysis of NRF2 and nuclear counterstain Hoechst. (F) NRF2 nuclear staining was quantified as the amount of cells that had clear NRF2 nuclear localization compared to the total amount of cells (n=~50 cells across 5–6 images were quantified).

Article Snippet: Antibodies were purchased from Santa Cruz Biotechnology (NRF2, KEAP1, NQO1, BRAF, GAPDH, p-ERK, ERK, SOX9), Cell Signaling Technology (Tubulin, FAM129B), MyBioSource (p-FAM129B), BD Biosciences (β-catenin), New England Biolabs (CBD), and Trevigen (HA).

Techniques: Transfection, Western Blot, Control, Immunofluorescence, Staining

Journal: Molecular carcinogenesis

Article Title: FAM129B-dependent activation of NRF2 promotes an invasive phenotype in BRAF mutant melanoma cells

doi: 10.1002/mc.23295

Figure Lengend Snippet: Constitutive activation of the MAPK signaling pathway via a mutation in BRAF (V600E) increases phosphorylation of FAM129B and mediates its cytosolic localization. In turn, FAM129B binds to KEAP1, preventing association between KEAP1 and NRF2, thus NRF2 protein accumulates, translocates to the nucleus, and activates transcription of its target genes.

Article Snippet: Antibodies were purchased from Santa Cruz Biotechnology (NRF2, KEAP1, NQO1, BRAF, GAPDH, p-ERK, ERK, SOX9), Cell Signaling Technology (Tubulin, FAM129B), MyBioSource (p-FAM129B), BD Biosciences (β-catenin), New England Biolabs (CBD), and Trevigen (HA).

Techniques: Activation Assay, Mutagenesis, Phospho-proteomics

Journal: Biomedicines

Article Title: Activation of MAP Kinase Pathway by Polyisoprenylated Cysteinyl Amide Inhibitors Causes Apoptosis and Disrupts Breast Cancer Cell Invasion

doi: 10.3390/biomedicines12030470

Figure Lengend Snippet: Proposed mechanism of action of the PCAIs in breast cancer cell lines. Abbreviations: RTK, Receptor Tyrosine Kinase; GRB2, Growth Factor Receptor Bound Protein 2; SOS, Son-of-Sevenless; GDP, Guanosine Diphosphate; GTP, Guanosine Triphosphate; RAS, Rat Sarcoma; BRAF, Rapidly Accelerated Fibrosarcoma, v-Raf Murine Sarcoma Viral Oncogene Homolog B; CRAF, RAF Proto-Oncogene Serine/Threonine-Protein Kinase; MEK, Mitogen-Activated Protein Kinase Kinase; ERK, Extracellular-Signal-Regulated Kinases; p90RSK, 90 kDa Ribosomal s6 Kinases; AKT, Protein Kinase B; BAK1, BCL2 Antagonist/Killer 1.

Article Snippet: Primary antibodies specific to phosphorylated proteins BRAF (p-BRAF, Cat #2696), CRAF (p-CRAF, Cat #9427), MEK 1/2 (p-MEK 1/2, Cat #9154S), ERK 1/2 (p-ERK1/2, Cat #4370S), p90RSK (p-p90RSK, Cat #11989S), AKT (p-AKT, Cat # 4060s), vinculin (E1E9V, Cat #13901), full-length Caspase 3 (Cat #9662S), full-length Caspase 7 (Cat #9492S), secondary antibodies anti-mouse IgG, HRP-linked antibody (Cat #7076), and anti-rabbit IgG, HRP-linked antibody (Cat #7074), and housekeeping protein GAPDH (Cat #5174S) or α-actinin (Cat #6478S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: Nature Communications

Article Title: Autophagy proteins regulate ERK phosphorylation

doi: 10.1038/ncomms3799

Figure Lengend Snippet: ( a ) EGF enhances colocalization of phosphorylated (P)-bRAF, P-MEK and P-ERK with LC3. Immunofluorescence (IF) showing colocalization of P-bRAF (green), P-MEK (green) and P-ERK (green) with LC3 (red) in NIH/3T3 cells in presence or absence of EGF (10 min as shown in scheme). The bars represent mean±s.e.m. ** P <0.01, *** P <0.001; Student’s t -test, 60 cells analysed from two experiments. Scale bars, 10 μm. ( b ) P-ERK colocalizes with pre-autophagosomal and autophagosomal structures. IF depicting colocalization of P-ERK (green or red) with LC3 (red), ATG5–ATG12 (red), ATG16 (green), vps34 (red), WIPI1 (red), WIPI2 (red) and P-ULK1 (red) in EGF-treated NIH/3T3 cells. Scale bars, 10 μm. The bars represent mean±s.e.m. 50 cells analysed from two experiments. ( c ) Growth factors in serum maintain P-ERK/LC3 colocalization. IF depicting colocalization of P-ERK (green) with LC3 (red) in 2 h serum-fed NIH/3T3 cells. ( a – c ) Native merged images or images with colocalization highlighted in white pixels are shown. Nuclei are blue (DAPI). Scale bar, 10 μm.

Article Snippet: Antibodies ( ) for ATG7, ATG4B, LC3B, P-bRAF (Ser445), bRAF, P-cRAF (Ser338), cRAF, P-p90RSK (Thr359/Ser363), P-MEK1/2 (Ser217/221), MEK1/2, P-ERK1/2 (Thr202/Tyr204) (rabbit and mouse species-specific), ERK1/2, ERK1, ERK2, P-Elk1 (Ser383), P-mTOR (Ser2448), mTOR and P-ULK1 (Ser555) were from Cell Signaling (Danvers, MA, USA); ATG5–ATG12 and ULK1 from Novus Biologicals (Littleton, CO, USA); p62 from Enzo life sciences (Plymouth Meeting, PA, USA); ATG16, Cathepsin B, Cathepsin L, GABARAP and GATE16 (sc-28938), P-c-jun (Ser63/73), c-jun, P-JNK (Thr183/Tyr185), JNK, IκBα, Rab7 and TFEB were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Beclin1 from BD Biosciences (San Jose, CA, USA); GAPDH, WIPI1 and WIPI2 antibodies from Abcam (Cambridge, MA, USA); Histone3 from Millipore (Billerica, MA, USA), β-actin from Sigma-Aldrich and JL-8 for CFP detection from Clontech (Mountain View, CA, USA).

Techniques: Immunofluorescence

Journal: Nature Communications

Article Title: Autophagy proteins regulate ERK phosphorylation

doi: 10.1038/ncomms3799

Figure Lengend Snippet: ( a ) EGF enhances nuclear LC3-II content in hepatocytes. Immunofluorescence (IF) depicting nuclear LC3-II in RALA hepatocytes in presence or absence of EGF (10 min). Native/inverted images are shown. Scale bar, 5 μm. Bars represent mean±s.e.m. *** P <0.001 compared with control (Con); Student’s t -test, 50 cells from two experiments. ( b ) EGF enhances nuclear P-ERK/LC3-II colocalization. IF depicting P-ERK (green)/LC3-II (red) colocalization in untreated (Con) or EGF-treated NIH/3T3 cells. Native images (top)/images with colocalization in white pixels (bottom) are shown. Scale bar, 5 μm. Bars represent mean±s.e.m. *** P <0.001 compared with Con; Student’s t -test, 50 cells from two experiments. ( c ) Adipogenic differentiation increases nuclear LC3-II. Images depict nuclear LC3-II in 3T3-L1 preadipocytes in maintenance or differentiation medium (2 h). Scale bar, 5 μm. Bars represent mean±s.e.m. ** P <0.01 compared with Con; Student’s t -test, 50 cells from two experiments. ( d ) Nuclear P-ERK/LC3-II colocalization in serum-fed cells. IF showing P-ERK (green)/LC3 (red) colocalization in 2 h serum-fed NIH/3T3 cells. Native images (top)/images with colocalization in white pixels (bottom) are shown. Scale bar, 5 μm. ( e ) LC3 interacts with ERK in vivo . Immunoblots showing co-immunoprecipitation of LC3 with ERK, MEK and bRAF in homogenate (Hom) ( e , top), and of LC3 with P- and total ERK in nuclear fractions from mouse livers ( e , bottom). ( f ) Blocking nuclear transport decreases EGF-induced increase in nuclear LC3-II. LC3 IF (red) in EGF-treated NIH/3T3 cells pre-exposed (30 min) or not to WGA. Bars represent mean±s.e.m. *** P <0.001 compared to with; Student’s t -test, 60 cells from n =3. Scale bar, 10 μm. ( g ) Blocking nuclear transport decreases nuclear ERK content. ERK IF (green) in EGF-treated NIH/3T3 cells pre-exposed (30 min) or not to WGA. Bars represent mean±s.e.m. *** P <0.001 compared with Con; Student’s t -test. ( h ) Blocking nuclear transport does not modify P-ERK/LC3-II colocalization. IF depicting nuclear P-ERK (green)/LC3 (red) colocalization in EGF-treated NIH/3T3 cells pre-exposed or not to WGA. For ( g ) and ( h ): Scale bar, 10 μm, bars are mean±s.e.m. 50 cells from n =2. Nuclei are blue (DAPI). Arrows indicate LC3 puncta, ERK or colocalization.

Article Snippet: Antibodies ( ) for ATG7, ATG4B, LC3B, P-bRAF (Ser445), bRAF, P-cRAF (Ser338), cRAF, P-p90RSK (Thr359/Ser363), P-MEK1/2 (Ser217/221), MEK1/2, P-ERK1/2 (Thr202/Tyr204) (rabbit and mouse species-specific), ERK1/2, ERK1, ERK2, P-Elk1 (Ser383), P-mTOR (Ser2448), mTOR and P-ULK1 (Ser555) were from Cell Signaling (Danvers, MA, USA); ATG5–ATG12 and ULK1 from Novus Biologicals (Littleton, CO, USA); p62 from Enzo life sciences (Plymouth Meeting, PA, USA); ATG16, Cathepsin B, Cathepsin L, GABARAP and GATE16 (sc-28938), P-c-jun (Ser63/73), c-jun, P-JNK (Thr183/Tyr185), JNK, IκBα, Rab7 and TFEB were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Beclin1 from BD Biosciences (San Jose, CA, USA); GAPDH, WIPI1 and WIPI2 antibodies from Abcam (Cambridge, MA, USA); Histone3 from Millipore (Billerica, MA, USA), β-actin from Sigma-Aldrich and JL-8 for CFP detection from Clontech (Mountain View, CA, USA).

Techniques: Immunofluorescence, Control, In Vivo, Western Blot, Immunoprecipitation, Blocking Assay