Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: ( a ) Multiple sequence alignment of metazoan Arl15 orthologues and human Arl5b. The conserved G1 (GxxxxGK[ST]) and G3 (ExGG[AGS]) motifs (DxGGQ for Arl5b) are underlined, and the switch-II region is marked. In human Arl15 sequence, R90, R95, and Y96 are indicated by #. The following UniProt sequences of Arl15 were used: anemone, A7SYP3; trichoplax, B3RRR0; limpet, V4BVB7; lamprey, S4R642; fish, A5PMK4; chicken, F1NMW9; frog, F6WKV0; human, Q9NXU5. The Uniprot ID of human Arl5b is Q96KC2. ( b ) Schematic diagrams showing the domain organization of Smad1, 2, 3, and 4 constructs. Numbers indicate corresponding amino acid positions in the full-length protein sequence. SAD, Smad4 activation domain. ( c ) The linker region of Smad4 does not interact with the GTP-mutant form of Arl15. Bead-immobilized GST-fusions of Smad4 fragments were incubated with HEK293T cell lysate expressing Arl15-AL-GFP or Arl15-TN-GFP, and pull-downs were immunoblotted against GFP. Loading of GST-fusions is shown below by Coomassie staining. 1, GST-Smad4; 2, GST-Smad4-MH2; 3, GST-Smad4-linker; 4, GST-Smad4-MH1; 5, GST. * indicates specific band. ( d ) Arl15 switch-II mutant, Arl15-AL-sw2-GFP, can bind to GTP. HEK293T cells lysates expressing indicated GFP-tagged proteins were incubated with the GTP-agarose, and pull-downs were immunoblotted against GFP. GFP-DEN(W34A), which is not a G protein, serves as a negative control. In ( c and d ), molecular weights (in kDa) are labeled in all immunoblots. Figure 1—figure supplement 1—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Sequencing, Construct, Activation Assay, Mutagenesis, Incubation, Expressing, Staining, Negative Control, Labeling, Western Blot

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: ( a ) The GTP-mutant form of Arl15 specifically immunoprecipitated endogenous Smad4. HEK293T cell lysates transiently expressing C-terminally GFP-tagged small G proteins were incubated with GFP-Trap beads, and immunoprecipitates were immunoblotted against Smad4 and GFP. 1 and 2 indicate Arl15-(WT, AL, or TN)-GFP and Arl5b-(WT, Q70L, or T30N)-GFP bands, respectively. Arl5b serves as a negative control. IP, immunoprecipitation. ( b ) Endogenous Smad4 immunoprecipitated Arl15 in the presence of GMPPNP, but not GDP. HEK293T cell lysates were incubated with anti-Smad4 antibody in the presence of 1 µM GMPPNP or GDP, and immunoprecipitates were immunoblotted against Arl15 and Smad4. ( c, d ) TGFβ1 stimulates the interaction between Smad4 and Arl15, and Arl15 promotes the interaction between Smad4 and phospho-Smad2/3. In ( c ), HEK293T cells subjected to Arl15 or control knockdown were serum-starved for 4 hr followed by further serum starvation or 5 ng/ml TGFβ1 treatment for 20 hr. Cell lysates were incubated with anti-Smad4 antibody, and immunoprecipitates were immunoblotted against indicated antibodies. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Under the control knockdown, the relative amount of Arl15 immunoprecipitated by Smad4 was quantified in ( d ). To calculate the relative amount of Arl15 immunoprecipitated by Smad4, the band intensity of immunoprecipitated Arl15 is divided by that of corresponding Smad4 and cell lysate Arl15, and the resulting value is further normalized by that of starvation. ( e, f ) The Smad4-MH2 domain specifically pulled down the GTP-mutant form of Arl15. In ( e ), bead-immobilized GST-fusions of Smad4 fragments were incubated with HEK293T cell lysate expressing Arl15-AL-GFP, and pull-downs were immunoblotted against GFP. 1, GST-Smad4; 2, GST-Smad4-linker-MH2; 3, GST-Smad4-MH2; 4, GST-Smad4-MH1; and 5, GST. * indicates specific band. The immunoblot is quantified in ( f ), in which the relative amount of Arl15-AL-GFP pulled down is calculated as the ratio of the intensity of the pull-down band to that of the cell lysate input band. ( g, h ) The MH2 domain of Smad4, but not that of Smad1, 2, and 3, directly interacts with the GTP-mutant form of Arl15. Bead-immobilized GST-fusions of MH2 domains were incubated with purified His-tagged Arl15-AL or TN, and pull-downs were immunoblotted against His-Tag. ( i ) The switch-II region of Arl15 is required for its interaction with Smad4. HEK293T cell lysates expressing indicated proteins were incubated with GFP antibody, and immunoprecipitates were immunoblotted against Myc-tag and GFP. In Arl15-AL-sw2-GFP, the switch-II region of Arl15-AL-GFP is replaced by that of Arl5b. ( j, k ) Cancer missense mutations in the MH2 domain of Smad4 can compromise Arl15-Smad4 interaction. In ( j ), HEK293T cell lysates expressing indicated GFP-Smad4 mutants were incubated with bead-immobilized GST-Arl15-AL, and pull-downs were immunoblotted for GFP. The result was quantified in ( k ). The relative amount of Smad4 mutant pulled down by GST-Arl15-AL is calculated by dividing the band intensity of GFP-Smad4 in pull-down by that in cell lysate, and the resulting value is further normalized by that of GFP-Smad4-WT. In ( e , g , h , and j ), the loading of fusion proteins was shown by Coomassie staining. In ( d , f , and k ), error bar, mean ± SD of n=3 experiments. p values are from the t -test (unpaired and two-tailed). NS, not significant (p>0.05). *, p≤0.05; *****, p≤0.000005. Red dot, individual data point. Molecular weights (in kDa) are labeled in all immunoblots. Figure 1—source data 1. Uncropped gel and blot images for . Chemiluminescence images are unboxed; black box, the cropped region shown in the corresponding figures; blue box, the white light image of pre-stained molecular weight marker bands (for immunoblots acquired by the CCD camera). Molecular weight (kDa) is labeled in all blots. For immunoblots acquired by film, molecular weight markers were manually traced. Figure 1—source data 2. Numerical data for graphs in . Figure 1—source data 3. List of positive hits from our yeast two-hybrid screening. The human kidney cDNA library (prey) was screened by Arl15-AL (bait). The table list all protein-coding clones from the screen. Figure 1—source data 4. List of SMAD4 missense cancer mutations that are tested in GST-Arl15-AL pull-down assay . Mutation information is from COSMIC. In the column ‘type of cancer identified’, the number of samples with the mutation is indicated in parenthesis. ‘Count’ displays the total number of samples with the mutation.

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Mutagenesis, Immunoprecipitation, Expressing, Incubation, Negative Control, Control, Knockdown, Western Blot, Purification, Staining, Two Tailed Test, Labeling, Molecular Weight, Marker, Two Hybrid Screening, cDNA Library Assay, Clone Assay, Pull Down Assay

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: HeLa cells under the normal culture condition were used. ( a, b ) Arl15 localizes to the Golgi and endolysosome. In ( a ), Arl15 localizes to the Golgi. Endogenous Arl15 and golgin245 (a Golgi marker) were stained by immunofluorescence in HeLa cells. Images were acquired by a wide-field microscope. In ( b ), Arl15-AL-GFP localizes to the PM and endolysosome. Live HeLa cells transiently co-expressing Arl15-AL-GFP and Lamp1-mCherry were imaged under a confocal microscope. Yellow arrows, colocalized puncta; cyan arrows, PM. ( c, d ) Smad4-MH2 displays better colocalization with the GTP-mutant form of Arl15 than full-length Smad4 at the endolysosome. Live HeLa cells co-expressing indicated mCherry and GFP-tagged proteins were imaged under a confocal microscope. Note that they colocalize at the endolysosome but not the Golgi. ( e ) Quantitative colocalization between Smad4-MH2 and various endolysosome markers. n=5 cells were imaged, and all red puncta (mCherry-Smad4-MH2 or Lamp1-mCherry) within each image were examined. Fractions of red puncta that visually colocalize with green puncta were calculated and plotted. Error bar, mean ± SD (n=5 cells). Red dot, individual data point. ( f ) Smad4-MH2 localizes to the EE, LE, and lysosome but not the RE. Live HeLa cells co-expressing indicated mCherry and GFP-tagged proteins were imaged under the confocal microscope. GFP-tagged 3×FYVE, TfR, Rab7, and Lamp1 are markers for the EE, RE, LE, and lysosome, respectively. Yellow arrows indicate colocalization. Scale bar, 10 µm. Figure 2—source data 1. Numerical data for the graph in .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Marker, Staining, Immunofluorescence, Microscopy, Expressing, Mutagenesis

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: HEK293T cells under the normal culture condition were used unless specified. ( a ) The GTP-mutant form of Arl15 specifically pulled down endogenous R-Smads in addition to Smad4. Bead-immobilized GST-fusion proteins were incubated with the cell lysate, and pull-downs and the cell lysate were immunoblotted against indicated Smads. 1, 2, and 3 indicate GST-Arl15 (AL or TN), GST-Arl5b (Q70L or T30N), and GST band. Arl5b serves as a negative control. ( b, c ) The GTP-mutant form of Arl15 pulled down more exogenously expressed Smad1 when Smad4 was co-expressed. In ( b ), bead-immobilized GST-fusion proteins were incubated with cell lysates expressing indicated proteins, and pull-downs and the cell lysates were immunoblotted against Smad1 and 4. 1, Flag-Smad1; 2, endogenous Smad1/5/8; 3, GST-Arl15 (AL or TN); 4, GST; 5, GFP-Smad4; *, the weak band of Flag-Smad1 that was pulled down without co-expression of Smad4. The percentage of Flag-Smad1 pulled down, calculated as the ratio of the intensity of the pull-down band to that of the corresponding 1% cell lysate input band, is plotted in ( c ). ( d, e, f ) The GTP-mutant form of Arl15 immunoprecipitated much less endogenous phospho-Smad2/3 upon Smad4 depletion. In ( d ), cells subjected to siRNA-mediated knockdown were transiently transfected to express Arl15-AL-GFP. After cells were treated with 5 ng/ml TGFβ1 for 20 hr, cell lysates were incubated with the anti-GFP antibody, and immunoprecipitated proteins were blotted together with cell lysates for indicated proteins. IP, immunoprecipitation. The quantification of blots is shown in ( e , f ). To calculate the relative Smad4 protein level in cell lysate, the band intensity of Smad4 in cell lysate is normalized by that of corresponding GAPDH, and the resulting value is further normalized by that of the control knockdown. To calculate the relative amount of phospho-Smad2/3 immunoprecipitated by Arl15-AL-GFP, the band intensity of immunoprecipitated phospho-Smad2/3 is normalized by that of corresponding Arl15-AL-GFP and cell lysate phospho-Smad2/3, and the resulting value is further normalized by that of the control knockdown. In ( c , e , and f ), error bar, mean ± SD of n=3 experiments. p values are from the t -test (unpaired and two-tailed). **, p≤0.005; ***, p≤0.0005. Red dot, individual data point. ( g ) Arl15-GTP, Smad4 and Smad2 can assemble into a complex. Bead-immobilized GST-fusion proteins were incubated with indicated purified His-tagged Smads, and pull-downs were immunoblotted against His-tag. 1, His-Smad4; 2, His-Smad2-SE; 3, GST-Arl15 (AL or TN); 4, GST. The loading of fusion proteins is shown by Coomassie staining in ( a , b , and g ). Molecular weights (in kDa) are labeled in all immunoblots and gels. Figure 3—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of . Figure 3—source data 2. Numerical data for graphs in .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Mutagenesis, Incubation, Negative Control, Expressing, Immunoprecipitation, Knockdown, Transfection, Control, Two Tailed Test, Purification, Staining, Labeling, Western Blot

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: HEK293T cells under the normal culture condition were used. ( a ) The anti-Smad2/3 mAb primarily detects endogenous Smad2 in HEK293T cells. Cells were subjected to GL2, Smad2, or Smad3 siRNA knockdown, and the resulting cell lysates were blotted for indicated proteins. Ratios of the intensity of Smad2/3 band to that of GAPDH band are displayed below. ( b ) The GTP-mutant form of Arl15 pulls down more exogenously expressed Smad2 when Smad4 was co-expressed. Bead-immobilized GST-fusion proteins were incubated with cell lysates expressing indicated proteins, and pull-downs and 1% cell lysate inputs were immunoblotted against Smad2 (anti-Smad2/3 mAb) and Smad4. In the middle panel, the same blot was sequentially blotted for Smad2 (upper blot) followed by Smad4 (lower blot). 1, GFP-Smad2; 2, GST-Arl15 (AL or TN); 3, GST; 4, GFP-Smad4; #, endogenous Smad2; *, the weak band of GFP-Smad2 that was pulled down without co-expression of Smad4. The normalized pull-down of GFP-Smad2, calculated by the ratio of the intensity of the pull-down band to that of 1% cell lysate input band, is plotted in the right panel. Loading of fusion proteins is shown by Coomassie staining. Molecular weights (in kDa) are labeled in immunoblots and gels. Figure 3—figure supplement 1—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Knockdown, Mutagenesis, Incubation, Expressing, Staining, Labeling, Western Blot

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: HEK293T cells under the normal culture condition were used. ( a ) The GTP-mutant form of Arl15 opens Smad4 by inhibiting the intramolecular interaction between the MH1 and MH2 domain of Smad4. Bead-immobilized GST-Smad4-MH1 was incubated with the cell lysates expressing indicated proteins, and pull-downs and the cell lysates were immunoblotted against HA or Myc-tag. ( b ) The normalized pull-down of HA-Smad4-MH2 for assays conducted in ( a ). The ratio of the intensity of the pull-down to that of the corresponding cell lysate band was calculated and plotted. ( c ) Arl15-GTP increases the intermolecular interaction between the Smad4-MH2 and Smad2-MH2 domain. Bead-immobilized GST-Smad2-MH1 domain was incubated with the cell lysates expressing indicated proteins, and pull-downs and the cell lysates were immunoblotted against indicated tags. ( d ) The normalized pull-down of Myc-Smad2-MH2 for assays conducted in ( c ). Quantification was the same as in ( b ). ( e, f ) Arl15-GTP promotes the interaction between Smad4 and phosphomimetic mutant of Smad2, Smad2-SE. In ( e ), bead-immobilized GST-Smad4 was incubated with the cell lysates expressing indicated proteins, and pull-downs and the cell lysates were immunoblotted against indicated tags or protein. 1, GFP-Smad2 (WT, SA or SE); 2, GFP; 3, endogenous Arl15 or overexpressed Arl15-AL. In ( f ), the cell lysates expressing indicated proteins were incubated with anti-GFP antibody, and the immunoprecipitates and the cell lysates were immunoblotted against indicated tags or protein. 1, Myc-Smad4; 2, endogenous Arl15 or overexpressed Arl15-AL; 3, GFP-Smad2 (WT, SA or SE); 4, GFP; #, non-specific band. ( g ) Arl15-GTP, Smad4, and Smad2-SE can assemble into a complex. Bead-immobilized GST-Arl15-AL was incubated with the cell lysates expressing indicated proteins, and pull-downs and the cell lysates were blotted. 1, GFP-Smad2 (WT, SA or SE); 2, GFP; 3, Myc-Smad4. In ( a , c , e and g ), loading of GST-fusion proteins is shown by Coomassie staining. Molecular weights (in kDa) are labeled in all immunoblots. ( h, i ) Quantification plots of , showing that Arl15 promotes the interaction between Smad4 and phospho-Smad2/3. In ( h ), to calculate the relative cellular phospho-Smad2/3, the band intensity of cell lysate phospho-Smad2/3 is normalized by that of corresponding GAPDH. In ( i ), to calculate the relative amount of phospho-Smad2/3 immunoprecipitated by Smad4, the band intensity of immunoprecipitated phospho-Smad2/3 is normalized by that of corresponding Smad4 and cell lysate phospho-Smad2/3, and the resulting value is further normalized by that of control knockdown. In ( b , d , h , and i ), error bar, mean ± SD of n=3 experiments. p values are from the t -test (unpaired and two-tailed). NS, not significant (p>0.05); *, p≤0.05; **, p≤0.005; ****, p≤0.00005. Red dot, individual data point. Figure 4—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of . Figure 4—source data 2. Numerical data for graphs in .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Mutagenesis, Incubation, Expressing, Staining, Labeling, Western Blot, Immunoprecipitation, Control, Knockdown, Two Tailed Test

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: HEK293T cells were cultured under normal condition. Bead-immobilized GST-Smad4 was incubated with cell lysates expressing indicated proteins, and pull-downs and the cell lysates were immunoblotted for GFP or Arl15. 1 and 2 indicate GFP-Smad1 (WT, SA, or SE) and GFP bands, respectively. Loading of fusion proteins is shown by Coomassie staining. Molecular weights (in kDa) are labeled in the immunoblot. Figure 4—figure supplement 1—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Cell Culture, Incubation, Expressing, Staining, Labeling, Western Blot

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: ( a–d ) Under the TGFβ1 treatment, phospho-Smad2/3, but not Arl15, translocated to the nucleus. In ( a ), serum-starved HeLa cells expressing GFP-golgin84 (a Golgi marker) and Arl15-WT-Myc were either further serum-starved or treated with 10 ng/ml TGFβ1 for 1 hr. Cells were stained for DNA (Hoechst 33342) and endogenous phospho-Smad2/3. The dotted line indicates the contour of the nucleus. Scale bar, 10 µm. In ( b ), HeLa cells were subjected to Arl15 or control knockdown. After being serum-starved for 4 hr, cells were either further serum-starved or treated with 10 ng/ml TGFβ1 for 1 hr. Total cell lysate (T) and cytosol (C) and nuclear (N) fractions were immunoblotted against indicated proteins. Molecular weights (in kDa) are labeled in immunoblots. Immunoblots were quantified in ( c and d ). In ( c ), the band intensity of phospho-Smad2/3 is first normalized by that of corresponding Nup133. To calculate the percentage of nuclear phospho-Smad2/3, the normalized nuclear phospho-Smad2/3 is divided by that of total cell lysate. In ( d ), the percentage of nuclear Smad4 is calculated as in ( c ). Red dot, individual data point. ( e ) Phospho-Smad2 (Smad2-SE) promotes the GAP activity of Smad4 toward Arl15. 40 µM GTP-loaded His-Arl15-WT or AL was incubated with 0.4 µM indicated His-Smads at 22 °C. Released inorganic phosphate was enzymatically converted and continuously monitored by absorbance at 360 nm. The absorbance was plotted against time. ( f ) The Smad4-MH2 domain possesses the GAP activity toward Arl15. The experiment was conducted as in ( e ). GST-fused Smad4 fragments were used. In ( c , d , e , and f ), error bar, mean ± SD of n=3 experiments. ( g, h, i ) Overexpression of GTP-form mutant of Arl15, Arl15-AL, inhibitsTGFβ1-stimulated nuclear translocation of phospho-Smad2/3 and Smad4. HeLa cells transiently expressing Arl15-AL or empty vector control (pCI-neo) were serum-starved for 4 hr followed by further starvation or 5 ng/ml TGFβ1 treatment for 20 hr. Nuclear fractionation, immunoblotting, and subsequent quantification are similar to ( b–d ). Figure 5—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of . Figure 5—source data 2. Numerical data for graphs in .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Expressing, Marker, Staining, Control, Knockdown, Labeling, Western Blot, Activity Assay, Incubation, Over Expression, Mutagenesis, Translocation Assay, Plasmid Preparation, Fractionation

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: ( a, b ) Arl15 is required for the efficient in vitro migration of MDA-MB-231 cells. MDA-MB-231 cells were subjected to lentivirus-transduced knockdown using indicated shRNA. When cells reached confluency, a strip of cells was scraped off, and the resulting gap was live-imaged to monitor the migration of cells. The percentage of the relative migration (see Materials and methods) is plotted in ( b ). ( c,d ) Arl15 is required for the efficient in vitro invasion and migration of MDA-MB-231 cells. MDA-MB-231 cells were subjected to lentivirus-transduced knockdown using indicated shRNA and were subsequently placed into cell culture filter chambers with basement matrix. Cells that invaded through the matrix and migrated to the lower surface of the filter were stained in ( c ). In ( d ), the relative invasion was calculated as described in Materials and methods. Scale bar, 100 µm. ( e, f ) Arl15 missense mutations identified from cancer patients compromise Arl15-Smad4 interaction and TGFβ signaling. In ( e ), ead-immobilized GST-Smad4 was incubated with HeLa cell lysates expressing Arl15-AL-Myc harboring indicated mutation, and pull-downs and the cell lysates were immunoblotted against Myc-tag. The normalized pull-down of Arl15-AL-Myc is shown below, and it is calculated as the ratio of the pull-down band intensity to the corresponding cell lysate band intensity. Loading of fusion proteins is shown by Coomassie staining. In ( f ), HeLa cells were co-transfected to express the dual-luciferase and Arl15-AL-Myc with indicated mutation. After 20 hr starvation, cells were subjected to the dual-luciferase assay and Western blot analysis for Myc-tag. In ( b , d , and f ), error bar, mean ± SD of n=3 experiments. p values were from the t -test (unpaired and two-tailed). *, p≤0.05; **, p≤0.005; ***, p≤0.0005; ******, p≤0.0000005. Red dot, individual data point. Molecular weights (in kDa) are labeled in all immunoblots. ( g ) A working model illustrating the molecular mechanism on how Arl15 regulates the TGFβ family signaling pathway. Smad2 is used as an example of the R-Smad. 2, Smad2; 4, Smad4; p, phosphate group at Smad2. See text for details. Figure 7—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of . Figure 7—source data 2. Numerical data for graphs in . Figure 7—source data 3. List of ARL15 missense cancer mutations that are tested in GST-Smad4 pull-down assay . Mutation information is from COSMIC. In the column “type of cancer identified”, the number of samples with the mutation is indicated in parenthesis. “Count” displays the total number of samples with the mutation. Figure 7—source data 4. List of ARL15 frameshift and nonsense mutations from COSMIC. In the column ‘type of cancer identified’, the number of samples with the mutation is indicated in parenthesis. ‘Count’ displays the total number of samples with the mutation.

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: In Vitro, Migration, Knockdown, shRNA, Stripping Membranes, Cell Culture, Staining, Incubation, Expressing, Mutagenesis, Transfection, Luciferase, Western Blot, Two Tailed Test, Labeling, Pull Down Assay

Journal: BMC Immunology

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen

doi: 10.1186/1471-2172-8-4

Figure Lengend Snippet: SDS-PAGE and Western Blot Analysis of Recombinant DEK. ( A ) Bacterial lysates and Ni-NTA column purified protein were diluted in reducing sample loading buffer, resolved by SDS-PAGE and stained with Coomassie blue. Lanes were loaded with 1) molecular weight (mw) marker, 2) control bacterial lysate, 10 μl, 3) pET-15b/DEK lysate, 10 μl, 4) 1.5 μg Ni-NTA column-purified DEK, 5) pET-15b/EGFP lysate, 10 μl, 6) 1.5 μg Ni-NTA column-purified EGFP. ( B ) Proteins were resolved by SDS-PAGE and then transferred to PVDF membrane using a Bis-Tris electrophoresis buffer system for western blot analysis. In blot (a) lanes contained: 1) mw marker, 2) control bacterial lysate, 3) pET-15b/DEK lysate, 10 μl, 4) 0.15 μg Ni-NTA column-purified DEK, 5) pET-15b/EGFP lysate, 10 μl, 6) 0.15 μg Ni-NTA column-purified EGFP. Blot (a) was probed with anti-6x-His antibody. For blot (b) a separate gel containing the same samples loaded in blot (a) was probed with anti-human DEK monoclonal antibody. Bound antibody was detected in both blots with alkaline phosphatase (AP)-conjugated rabbit anti-mouse IgG (H+L) secondary antibody, as described in Methods. Data is representative of more than three separate experiments.

Article Snippet: Blots were probed with anti-human DEK (BD Biosciences) and anti-His antibody (Serotec, Raleigh, MC) at a 1:1000 dilution, followed by alkaline phosphatase conjugated rabbit anti-mouse IgG (H+L) (Abcam) at a 1:2500 dilution.

Techniques: SDS Page, Western Blot, Recombinant, Purification, Staining, Molecular Weight, Marker, Electrophoresis

Journal: BMC Immunology

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen

doi: 10.1186/1471-2172-8-4

Figure Lengend Snippet: Immunofluorescence (IF) Analysis of DEK expression. AGN2a (column 2) or AGN2a/DEK (columns 1 and 3) were plated on glass chamber slides, fixed with 4% paraformaldehyde and visualized using a Nuance multispectral imaging system (Nuance 1P46, Cambridge Research and Instrumentation, Inc. Woburn, MA) mounted on a Zeiss Axio Imager Z1 microscope. Image files (200×) were captured and merged using the Axiovision 4.5 package (open program). For all cells, nuclei were stained blue with DAPI. Cells were stained with anti-human DEK antibody (1:100, columns 1 and 2) or similarly diluted isotype control antibody (column 3). Bound antibody was detected with goat anti-mouse IgG (H+L) conjugated to Alexa Fluor 555. Row (A) shows the merged DAPI and Alexa Fluor 555 image, and row (B) shows the staining of the Alexa Fluor 555 image alone. Data is representative of more than three separate experiments.

Article Snippet: Blots were probed with anti-human DEK (BD Biosciences) and anti-His antibody (Serotec, Raleigh, MC) at a 1:1000 dilution, followed by alkaline phosphatase conjugated rabbit anti-mouse IgG (H+L) (Abcam) at a 1:2500 dilution.

Techniques: Immunofluorescence, Expressing, Imaging, Microscopy, Staining

Journal: BMC Immunology

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen

doi: 10.1186/1471-2172-8-4

Figure Lengend Snippet: ELISA Analysis of Immune Sera for DEK Reactivity. 96-well plates were coated with recombinant DEK or EGFP (10 μg/ml) produced in bacterial vectors. Wells were blocked, and 100 μl of test serum (as described on the x-axis), diluted 1:100, was added to each well. Sera used for this assay was the same serum used for cDNA library screening, and was pooled from 5 immunized mice. Serum from naïve A/J mice, mice treated with PC61 alone, or AGN2a-CD80/CD86 vaccine alone was derived from parallel experimental groups used in the immunization protocol. Following incubation at 37°C for 1 hour, secondary rabbit anti-mouse IgG (H+L) was used to detect bound antibody, as detailed in Methods. Also included was anti-human DEK monoclonal antibody (Anti-DEK-MAb) used at 0.5 μg/ml. Error bars show standard deviations for triplicate sample wells in the same assay.

Article Snippet: Blots were probed with anti-human DEK (BD Biosciences) and anti-His antibody (Serotec, Raleigh, MC) at a 1:1000 dilution, followed by alkaline phosphatase conjugated rabbit anti-mouse IgG (H+L) (Abcam) at a 1:2500 dilution.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Produced, cDNA Library Assay, Derivative Assay, Incubation

Journal:

Article Title: Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription

doi: 10.1093/nar/gkh195

Figure Lengend Snippet: Dnmt3a binds to Ubc9, PIAS1 and PIASxα in a yeast two-hybrid screen. Full-length Dnmt3a was fused to the GAL4-DBD and used as bait in a yeast two-hybrid screen against a human fetal liver cDNA library in AH109 yeast cells under high stringency selection conditions. Clones scoring positive for growth on selective media were confirmed using a beta-galactosidase assay in Y187 yeast cells, then sequenced. The identity of two representative, independently isolated clones is shown below the graph of beta-galactosidase activity (as relative units). The positive control (+) is the interaction between p53 and the SV40 T antigen, the negative control (–) is the interaction between p53 and lamin B, and ‘bait only’ is the GAL4-Dnmt3a construct alone under the appropriate selection conditions. Expression of full-length Dnmt3a in AH109 cells was confirmed by western blotting (data not shown). Values are the mean of three independent experiments and the error bar is the standard deviation from the mean.

Article Snippet: Antibodies used are as follows: mouse anti-GAL4-DBD RK5C1 (Santa Cruz Biotechnology, 1:1000 for western), rabbit anti-GAL4-DBD sc-577 (Santa Cruz Biotechnology, 1:500 for western), goat anti-Dnmt3a P-16 (Santa Cruz Biotechnology, 1:500 for western), rabbit anti-HDAC1 and HDAC2 (Affinity Bioreagents, 1:2000 for western), mouse anti-FLAG M2 (Sigma, 1:1000 for western), mouse anti-GFP 11E5 (Q Biogene, 1:1000 for western), goat anti-PIAS1/3 N-18 (Santa Cruz Biotechnology, 1:500 for western), and mouse anti-SUMO-1 (Zymed Laboratories, 1:1000 for western).

Techniques: Two Hybrid Screening, cDNA Library Assay, Selection, Clone Assay, β-Gal Assay, Isolation, Activity Assay, Positive Control, Negative Control, Construct, Expressing, Western Blot, Standard Deviation

Journal:

Article Title: Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription

doi: 10.1093/nar/gkh195

Figure Lengend Snippet: Ubc9, PIAS1 and PIASxα interact with Dnmt3a via a region within the N-terminal regulatory domain. A schematic diagram of the Dnmt3a protein is shown at the top with the PWWP, PHD and catalytic domains indicated. Each of the Dnmt3a deletion mutants, fused to the GAL4-DBD, was co-transformed with GAL4-AD fusions of Ubc9 (left), PIAS1 (middle), or PIASxα (right) into AH109 yeast cells. Thick lines denote the regions of Dnmt3a that were fused to the GAL4-DBD. Three colonies growing under high stringency selection for each transformation were selected, grown in liquid culture, then used for a beta-galactosidase assay (relative units). Graphs show the mean of three independent experiments (colonies) relative to the full-length Dnmt3a set at 100%. Error bars are the standard deviation from the mean.

Article Snippet: Antibodies used are as follows: mouse anti-GAL4-DBD RK5C1 (Santa Cruz Biotechnology, 1:1000 for western), rabbit anti-GAL4-DBD sc-577 (Santa Cruz Biotechnology, 1:500 for western), goat anti-Dnmt3a P-16 (Santa Cruz Biotechnology, 1:500 for western), rabbit anti-HDAC1 and HDAC2 (Affinity Bioreagents, 1:2000 for western), mouse anti-FLAG M2 (Sigma, 1:1000 for western), mouse anti-GFP 11E5 (Q Biogene, 1:1000 for western), goat anti-PIAS1/3 N-18 (Santa Cruz Biotechnology, 1:500 for western), and mouse anti-SUMO-1 (Zymed Laboratories, 1:1000 for western).

Techniques: Transformation Assay, Selection, β-Gal Assay, Standard Deviation

Journal:

Article Title: Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription

doi: 10.1093/nar/gkh195

Figure Lengend Snippet: Dnmt3a is sumoylated in vivo and in vitro. (A) GAL4-DBD-tagged Dnmt3a (2 µg) was co-transfected with or without a SUMO-1 expression vector (1.5 µg) into COS-7 cells. Whole cell extracts (WCE) were prepared in the presence of NEM and the migration position of the transfected Dnmt3a determined by western blotting with an anti-GAL4-DBD antibody (left panel). Dnmt3a was also immunoprecipitated from the transfected WCEs with a rabbit polyclonal GAL4-DBD antibody (‘r’), and the molecular weight of the total pool of GAL4-Dnmt3a determined by western blotting with a mouse monoclonal GAL4-DBD antibody (‘m’, middle panel). The presence of slower migrating forms of Dnmt3a in the ‘+SUMO-1’ lane indicates that Dnmt3a is sumoylated. When the blot is stripped and re-probed with a SUMO-1 antibody, only the slower migrating species are detectable and these higher molecular weight forms are specifically enriched in the ‘+SUMO-1’ reaction (right panel). (B) Dnmt3a is sumoylated in vitro. In vitro sumoylation reactions were carried out in the presence of 35S-labeled in vitro transcribed/translated (IVT) Dnmt3a, 200 ng recombinant SAE1/SAE2, 1 µg Ubc9, 2 mM ATP, ATP regeneration system, and 10 µg of SUMO-1 or 5 µg GST-SUMO-1 as indicated at the top of each panel. Molecular weight markers (kDa), are indicated at the left. The open arrow denotes the position of the unmodified GAL4-Dnmt3a and the filled arrow shows the migration position of the sumo-modified Dnmt3a.

Article Snippet: Antibodies used are as follows: mouse anti-GAL4-DBD RK5C1 (Santa Cruz Biotechnology, 1:1000 for western), rabbit anti-GAL4-DBD sc-577 (Santa Cruz Biotechnology, 1:500 for western), goat anti-Dnmt3a P-16 (Santa Cruz Biotechnology, 1:500 for western), rabbit anti-HDAC1 and HDAC2 (Affinity Bioreagents, 1:2000 for western), mouse anti-FLAG M2 (Sigma, 1:1000 for western), mouse anti-GFP 11E5 (Q Biogene, 1:1000 for western), goat anti-PIAS1/3 N-18 (Santa Cruz Biotechnology, 1:500 for western), and mouse anti-SUMO-1 (Zymed Laboratories, 1:1000 for western).

Techniques: In Vivo, In Vitro, Transfection, Expressing, Plasmid Preparation, Migration, Western Blot, Immunoprecipitation, Molecular Weight, Labeling, Recombinant, Modification

Journal:

Article Title: Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription

doi: 10.1093/nar/gkh195

Figure Lengend Snippet: PIAS1 and PIASxα do not enhance sumoylation of Dnmt3a in vivo or in vitro. (A) Two micrograms of full-length GAL4-Dnmt3a was transfected without (lane 1) or with 1.5 µg of SUMO-1 expression vector (lane 2) into COS-7 cells and whole cell extracts were prepared for western analysis with anti-GAL4-DBD antibody. In lanes 3 and 4, four micrograms of PIAS1 or PIASxα expression vector, respectively, are co-transfected with the SUMO-1 and GAL4-Dnmt3a expression vectors. (B) In vitro sumoylation reactions containing 35S-labeled-IVT Dnmt3a, performed as described in Figure ​Figure5B,5B, were supplemented with recombinant GST-PIASxα (1, 5, 10 µg) and the reaction products analyzed on a 6% SDS–PAGE gel.

Article Snippet: Antibodies used are as follows: mouse anti-GAL4-DBD RK5C1 (Santa Cruz Biotechnology, 1:1000 for western), rabbit anti-GAL4-DBD sc-577 (Santa Cruz Biotechnology, 1:500 for western), goat anti-Dnmt3a P-16 (Santa Cruz Biotechnology, 1:500 for western), rabbit anti-HDAC1 and HDAC2 (Affinity Bioreagents, 1:2000 for western), mouse anti-FLAG M2 (Sigma, 1:1000 for western), mouse anti-GFP 11E5 (Q Biogene, 1:1000 for western), goat anti-PIAS1/3 N-18 (Santa Cruz Biotechnology, 1:500 for western), and mouse anti-SUMO-1 (Zymed Laboratories, 1:1000 for western).

Techniques: In Vivo, In Vitro, Transfection, Expressing, Plasmid Preparation, Western Blot, Labeling, Recombinant, SDS Page

Journal:

Article Title: Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription

doi: 10.1093/nar/gkh195

Figure Lengend Snippet: Sumoylation differentially affects several known protein–protein interactions involving Dnmt3a. (A) HCT116 cells were transfected for 24 h with 2.0 µg of each of the expression constructs listed along the top. Whole cell extracts were prepared and subjected to immunoprecipitation with a mouse anti-GAL4-DBD tag antibody to pull-down Dnmt3a. Subsequent western blotting with antibodies against HDAC1 (left panel), or HDAC2 (right panel), revealed that Dnmt3a interacts with both HDAC1 and HDAC2 and that this interaction is markedly reduced under conditions favoring sumoylation of Dnmt3a (co-transfection of 1.5 µg of SUMO-1 expression vector). Levels of HDAC1/2 and GAL4-Dnmt3a were not affected by SUMO-1 over-expression (lower two panels, ‘Inputs’). (B) HCT116 cells were transfected with tagged Dnmt3a and Dnmt3b1 and whole cell extracts prepared as described above. Immunoprecipitations were carried out with anti-GAL4-DBD antibody and western blotting of the bound material was performed with an anti-FLAG antibody to detect the presence of Dnmt3b1. SUMO-1 expression did not alter the ability of Dnmt3a to interact with Dnmt3b1 (upper panel), nor did it affect the levels of the transfected proteins (lower two panels, ‘Inputs’). Similar results were obtained using COS-7 cells (not shown).

Article Snippet: Antibodies used are as follows: mouse anti-GAL4-DBD RK5C1 (Santa Cruz Biotechnology, 1:1000 for western), rabbit anti-GAL4-DBD sc-577 (Santa Cruz Biotechnology, 1:500 for western), goat anti-Dnmt3a P-16 (Santa Cruz Biotechnology, 1:500 for western), rabbit anti-HDAC1 and HDAC2 (Affinity Bioreagents, 1:2000 for western), mouse anti-FLAG M2 (Sigma, 1:1000 for western), mouse anti-GFP 11E5 (Q Biogene, 1:1000 for western), goat anti-PIAS1/3 N-18 (Santa Cruz Biotechnology, 1:500 for western), and mouse anti-SUMO-1 (Zymed Laboratories, 1:1000 for western).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, Western Blot, Cotransfection, Plasmid Preparation, Over Expression

Journal:

Article Title: Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription

doi: 10.1093/nar/gkh195

Figure Lengend Snippet: SUMO-1 modification of Dnmt3a eliminates its capacity to repress transcription. (A) Full-length Dnmt3a represses transcription when fused to the GAL4-DBD. Increasing amounts (indicated by the black wedge) of GAL4-Dnmt3a (0.5, 1.0, 2.5, 5.0 µg) or untethered Dnmt3a (0.5, 1.0, 2.5, 5.0 µg) were co-transfected into HCT116 cells along with 10 ng of an SV40-Renilla luciferase construct (to control for transfection efficiency), and 1.0 µg of a firefly luciferase reporter gene driven by five GAL4 DNA binding sites. Both firefly and Renilla luciferase activities were measured from the same whole cell extract preparation after 24 h using the Promega Dual Luciferase Assay kit. (B) GAL4-Dnmt3a (4.0 µg) was co-transfected with increasing amounts of SUMO-1 expression vector (1.0, 2.5, 5.0, 8.0 µg) into HCT116 cells. Transfection of equivalent amounts of SUMO-1 expression vector in the absence of GAL4-Dnmt3a did not alter promoter activity. (C) Effects of PIAS proteins on Dnmt3a transcriptional repression. GAL4-Dnmt3a (4.0 µg) was co-transfected with increasing amounts of PIASxα or PIAS1 expression vectors (1.0, 2.5, 5.0, 8.0 µg) or a mutant form of PIAS1 (Δ341–536, 2.5, 5.0, 8.0 µg) deleted for the RING-finger-like domain, into HCT116 cells and reporter activity determined after 24 h. Transfection of PIAS1 or PIASxα (2.5, 5.0, 8.0 µg) expression plasmids in the absence of GAL4-Dnmt3a did not affect reporter gene activity. All values were normalized for transfection efficiency (firefly luciferase/Renilla luciferase) and then set relative to the reporter activity of the 5× GAL4-BS-luciferase construct alone set at 100% (first bar in each series). Values are the average of two independent experiments and error bars are the range. Total DNA content was kept constant in each transfection by addition of ‘empty’ parental expression vector.

Article Snippet: Antibodies used are as follows: mouse anti-GAL4-DBD RK5C1 (Santa Cruz Biotechnology, 1:1000 for western), rabbit anti-GAL4-DBD sc-577 (Santa Cruz Biotechnology, 1:500 for western), goat anti-Dnmt3a P-16 (Santa Cruz Biotechnology, 1:500 for western), rabbit anti-HDAC1 and HDAC2 (Affinity Bioreagents, 1:2000 for western), mouse anti-FLAG M2 (Sigma, 1:1000 for western), mouse anti-GFP 11E5 (Q Biogene, 1:1000 for western), goat anti-PIAS1/3 N-18 (Santa Cruz Biotechnology, 1:500 for western), and mouse anti-SUMO-1 (Zymed Laboratories, 1:1000 for western).

Techniques: Modification, Transfection, Luciferase, Construct, Binding Assay, Expressing, Plasmid Preparation, Activity Assay, Mutagenesis

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: ( a ) The GTP-mutant form of Arl15 specifically immunoprecipitated endogenous Smad4. HEK293T cell lysates transiently expressing C-terminally GFP-tagged small G proteins were incubated with GFP-Trap beads, and immunoprecipitates were immunoblotted against Smad4 and GFP. 1 and 2 indicate Arl15-(WT, AL, or TN)-GFP and Arl5b-(WT, Q70L, or T30N)-GFP bands, respectively. Arl5b serves as a negative control. IP, immunoprecipitation. ( b ) Endogenous Smad4 immunoprecipitated Arl15 in the presence of GMPPNP, but not GDP. HEK293T cell lysates were incubated with anti-Smad4 antibody in the presence of 1 µM GMPPNP or GDP, and immunoprecipitates were immunoblotted against Arl15 and Smad4. ( c, d ) TGFβ1 stimulates the interaction between Smad4 and Arl15, and Arl15 promotes the interaction between Smad4 and phospho-Smad2/3. In ( c ), HEK293T cells subjected to Arl15 or control knockdown were serum-starved for 4 hr followed by further serum starvation or 5 ng/ml TGFβ1 treatment for 20 hr. Cell lysates were incubated with anti-Smad4 antibody, and immunoprecipitates were immunoblotted against indicated antibodies. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Under the control knockdown, the relative amount of Arl15 immunoprecipitated by Smad4 was quantified in ( d ). To calculate the relative amount of Arl15 immunoprecipitated by Smad4, the band intensity of immunoprecipitated Arl15 is divided by that of corresponding Smad4 and cell lysate Arl15, and the resulting value is further normalized by that of starvation. ( e, f ) The Smad4-MH2 domain specifically pulled down the GTP-mutant form of Arl15. In ( e ), bead-immobilized GST-fusions of Smad4 fragments were incubated with HEK293T cell lysate expressing Arl15-AL-GFP, and pull-downs were immunoblotted against GFP. 1, GST-Smad4; 2, GST-Smad4-linker-MH2; 3, GST-Smad4-MH2; 4, GST-Smad4-MH1; and 5, GST. * indicates specific band. The immunoblot is quantified in ( f ), in which the relative amount of Arl15-AL-GFP pulled down is calculated as the ratio of the intensity of the pull-down band to that of the cell lysate input band. ( g, h ) The MH2 domain of Smad4, but not that of Smad1, 2, and 3, directly interacts with the GTP-mutant form of Arl15. Bead-immobilized GST-fusions of MH2 domains were incubated with purified His-tagged Arl15-AL or TN, and pull-downs were immunoblotted against His-Tag. ( i ) The switch-II region of Arl15 is required for its interaction with Smad4. HEK293T cell lysates expressing indicated proteins were incubated with GFP antibody, and immunoprecipitates were immunoblotted against Myc-tag and GFP. In Arl15-AL-sw2-GFP, the switch-II region of Arl15-AL-GFP is replaced by that of Arl5b. ( j, k ) Cancer missense mutations in the MH2 domain of Smad4 can compromise Arl15-Smad4 interaction. In ( j ), HEK293T cell lysates expressing indicated GFP-Smad4 mutants were incubated with bead-immobilized GST-Arl15-AL, and pull-downs were immunoblotted for GFP. The result was quantified in ( k ). The relative amount of Smad4 mutant pulled down by GST-Arl15-AL is calculated by dividing the band intensity of GFP-Smad4 in pull-down by that in cell lysate, and the resulting value is further normalized by that of GFP-Smad4-WT. In ( e , g , h , and j ), the loading of fusion proteins was shown by Coomassie staining. In ( d , f , and k ), error bar, mean ± SD of n=3 experiments. p values are from the t -test (unpaired and two-tailed). NS, not significant (p>0.05). *, p≤0.05; *****, p≤0.000005. Red dot, individual data point. Molecular weights (in kDa) are labeled in all immunoblots. Figure 1—source data 1. Uncropped gel and blot images for . Chemiluminescence images are unboxed; black box, the cropped region shown in the corresponding figures; blue box, the white light image of pre-stained molecular weight marker bands (for immunoblots acquired by the CCD camera). Molecular weight (kDa) is labeled in all blots. For immunoblots acquired by film, molecular weight markers were manually traced. Figure 1—source data 2. Numerical data for graphs in . Figure 1—source data 3. List of positive hits from our yeast two-hybrid screening. The human kidney cDNA library (prey) was screened by Arl15-AL (bait). The table list all protein-coding clones from the screen. Figure 1—source data 4. List of SMAD4 missense cancer mutations that are tested in GST-Arl15-AL pull-down assay . Mutation information is from COSMIC. In the column ‘type of cancer identified’, the number of samples with the mutation is indicated in parenthesis. ‘Count’ displays the total number of samples with the mutation.

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Mutagenesis, Immunoprecipitation, Expressing, Incubation, Negative Control, Control, Knockdown, Western Blot, Purification, Staining, Two Tailed Test, Labeling, Molecular Weight, Marker, Two Hybrid Screening, cDNA Library Assay, Clone Assay, Pull Down Assay

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: HEK293T cells under the normal culture condition were used unless specified. ( a ) The GTP-mutant form of Arl15 specifically pulled down endogenous R-Smads in addition to Smad4. Bead-immobilized GST-fusion proteins were incubated with the cell lysate, and pull-downs and the cell lysate were immunoblotted against indicated Smads. 1, 2, and 3 indicate GST-Arl15 (AL or TN), GST-Arl5b (Q70L or T30N), and GST band. Arl5b serves as a negative control. ( b, c ) The GTP-mutant form of Arl15 pulled down more exogenously expressed Smad1 when Smad4 was co-expressed. In ( b ), bead-immobilized GST-fusion proteins were incubated with cell lysates expressing indicated proteins, and pull-downs and the cell lysates were immunoblotted against Smad1 and 4. 1, Flag-Smad1; 2, endogenous Smad1/5/8; 3, GST-Arl15 (AL or TN); 4, GST; 5, GFP-Smad4; *, the weak band of Flag-Smad1 that was pulled down without co-expression of Smad4. The percentage of Flag-Smad1 pulled down, calculated as the ratio of the intensity of the pull-down band to that of the corresponding 1% cell lysate input band, is plotted in ( c ). ( d, e, f ) The GTP-mutant form of Arl15 immunoprecipitated much less endogenous phospho-Smad2/3 upon Smad4 depletion. In ( d ), cells subjected to siRNA-mediated knockdown were transiently transfected to express Arl15-AL-GFP. After cells were treated with 5 ng/ml TGFβ1 for 20 hr, cell lysates were incubated with the anti-GFP antibody, and immunoprecipitated proteins were blotted together with cell lysates for indicated proteins. IP, immunoprecipitation. The quantification of blots is shown in ( e , f ). To calculate the relative Smad4 protein level in cell lysate, the band intensity of Smad4 in cell lysate is normalized by that of corresponding GAPDH, and the resulting value is further normalized by that of the control knockdown. To calculate the relative amount of phospho-Smad2/3 immunoprecipitated by Arl15-AL-GFP, the band intensity of immunoprecipitated phospho-Smad2/3 is normalized by that of corresponding Arl15-AL-GFP and cell lysate phospho-Smad2/3, and the resulting value is further normalized by that of the control knockdown. In ( c , e , and f ), error bar, mean ± SD of n=3 experiments. p values are from the t -test (unpaired and two-tailed). **, p≤0.005; ***, p≤0.0005. Red dot, individual data point. ( g ) Arl15-GTP, Smad4 and Smad2 can assemble into a complex. Bead-immobilized GST-fusion proteins were incubated with indicated purified His-tagged Smads, and pull-downs were immunoblotted against His-tag. 1, His-Smad4; 2, His-Smad2-SE; 3, GST-Arl15 (AL or TN); 4, GST. The loading of fusion proteins is shown by Coomassie staining in ( a , b , and g ). Molecular weights (in kDa) are labeled in all immunoblots and gels. Figure 3—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of . Figure 3—source data 2. Numerical data for graphs in .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Mutagenesis, Incubation, Negative Control, Expressing, Immunoprecipitation, Knockdown, Transfection, Control, Two Tailed Test, Purification, Staining, Labeling, Western Blot

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: HEK293T cells under the normal culture condition were used. ( a ) The anti-Smad2/3 mAb primarily detects endogenous Smad2 in HEK293T cells. Cells were subjected to GL2, Smad2, or Smad3 siRNA knockdown, and the resulting cell lysates were blotted for indicated proteins. Ratios of the intensity of Smad2/3 band to that of GAPDH band are displayed below. ( b ) The GTP-mutant form of Arl15 pulls down more exogenously expressed Smad2 when Smad4 was co-expressed. Bead-immobilized GST-fusion proteins were incubated with cell lysates expressing indicated proteins, and pull-downs and 1% cell lysate inputs were immunoblotted against Smad2 (anti-Smad2/3 mAb) and Smad4. In the middle panel, the same blot was sequentially blotted for Smad2 (upper blot) followed by Smad4 (lower blot). 1, GFP-Smad2; 2, GST-Arl15 (AL or TN); 3, GST; 4, GFP-Smad4; #, endogenous Smad2; *, the weak band of GFP-Smad2 that was pulled down without co-expression of Smad4. The normalized pull-down of GFP-Smad2, calculated by the ratio of the intensity of the pull-down band to that of 1% cell lysate input band, is plotted in the right panel. Loading of fusion proteins is shown by Coomassie staining. Molecular weights (in kDa) are labeled in immunoblots and gels. Figure 3—figure supplement 1—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Knockdown, Mutagenesis, Incubation, Expressing, Staining, Labeling, Western Blot

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: HEK293T cells under the normal culture condition were used. ( a ) The GTP-mutant form of Arl15 opens Smad4 by inhibiting the intramolecular interaction between the MH1 and MH2 domain of Smad4. Bead-immobilized GST-Smad4-MH1 was incubated with the cell lysates expressing indicated proteins, and pull-downs and the cell lysates were immunoblotted against HA or Myc-tag. ( b ) The normalized pull-down of HA-Smad4-MH2 for assays conducted in ( a ). The ratio of the intensity of the pull-down to that of the corresponding cell lysate band was calculated and plotted. ( c ) Arl15-GTP increases the intermolecular interaction between the Smad4-MH2 and Smad2-MH2 domain. Bead-immobilized GST-Smad2-MH1 domain was incubated with the cell lysates expressing indicated proteins, and pull-downs and the cell lysates were immunoblotted against indicated tags. ( d ) The normalized pull-down of Myc-Smad2-MH2 for assays conducted in ( c ). Quantification was the same as in ( b ). ( e, f ) Arl15-GTP promotes the interaction between Smad4 and phosphomimetic mutant of Smad2, Smad2-SE. In ( e ), bead-immobilized GST-Smad4 was incubated with the cell lysates expressing indicated proteins, and pull-downs and the cell lysates were immunoblotted against indicated tags or protein. 1, GFP-Smad2 (WT, SA or SE); 2, GFP; 3, endogenous Arl15 or overexpressed Arl15-AL. In ( f ), the cell lysates expressing indicated proteins were incubated with anti-GFP antibody, and the immunoprecipitates and the cell lysates were immunoblotted against indicated tags or protein. 1, Myc-Smad4; 2, endogenous Arl15 or overexpressed Arl15-AL; 3, GFP-Smad2 (WT, SA or SE); 4, GFP; #, non-specific band. ( g ) Arl15-GTP, Smad4, and Smad2-SE can assemble into a complex. Bead-immobilized GST-Arl15-AL was incubated with the cell lysates expressing indicated proteins, and pull-downs and the cell lysates were blotted. 1, GFP-Smad2 (WT, SA or SE); 2, GFP; 3, Myc-Smad4. In ( a , c , e and g ), loading of GST-fusion proteins is shown by Coomassie staining. Molecular weights (in kDa) are labeled in all immunoblots. ( h, i ) Quantification plots of , showing that Arl15 promotes the interaction between Smad4 and phospho-Smad2/3. In ( h ), to calculate the relative cellular phospho-Smad2/3, the band intensity of cell lysate phospho-Smad2/3 is normalized by that of corresponding GAPDH. In ( i ), to calculate the relative amount of phospho-Smad2/3 immunoprecipitated by Smad4, the band intensity of immunoprecipitated phospho-Smad2/3 is normalized by that of corresponding Smad4 and cell lysate phospho-Smad2/3, and the resulting value is further normalized by that of control knockdown. In ( b , d , h , and i ), error bar, mean ± SD of n=3 experiments. p values are from the t -test (unpaired and two-tailed). NS, not significant (p>0.05); *, p≤0.05; **, p≤0.005; ****, p≤0.00005. Red dot, individual data point. Figure 4—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of . Figure 4—source data 2. Numerical data for graphs in .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Mutagenesis, Incubation, Expressing, Staining, Labeling, Western Blot, Immunoprecipitation, Control, Knockdown, Two Tailed Test

Journal: eLife

Article Title: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex

doi: 10.7554/eLife.76146

Figure Lengend Snippet: ( a ) The Arl15-AL-induced transcription of SBE ×4 luc requires the kinase activity of the TGFβ type I receptor. HeLa cells co-expressing SBE ×4-driven firefly luciferase and SV40-driven renilla luciferase together with Arl15-AL or pBluescript SK vector DNA (control) were serum-starved for 4 hr followed by 20 hr treatment with starvation medium with or without 2 μM SB431542. The relative luciferase activities were subsequently acquired and normalized. ( b ) Arl15 positively regulates the BMP signaling pathway since overexpression of Arl15-WT or AL, but not TN, promotes the transcription of BRE-luc reporter. HeLa cells co-expressing BRE-driven firefly luciferase and SV40-driven renilla luciferase together with indicated Arl15 mutant, or pBluescript SK vector DNA (control) were serum-starved for 24 hr. The relative luciferase activities were subsequently acquired and normalized. ( c ) Endogenous Arl15 can be depleted by lentivirus-transduced shRNA knockdown. HeLa cells were lysed after lentivirus-transduced knockdown using indicated shRNA, and the resulting cell lysates were immunoblotted for Arl15 and GAPDH. ( d ) Arl15 is essential for efficient BMP signaling since its depletion reduces BMP2-stimulated transcription of BRE-luc reporter. After lentivirus-transduced knockdown of Arl15, HeLa cells expressing the dual-luciferase as described in ( b ) were serum-starved for 4 hr followed by 20 hr treatment with starvation medium supplemented with or without 100 ng/ml BMP2. The relative luciferase activities were subsequently acquired and normalized. ( e, f ) The ER-to-Golgi transport of ManII does not require Arl15. In ( e ), after lentivirus-transduced knockdown of Arl15, HeLa cells transiently expressing RUSH reporter ManII-SBP-GFP were subjected to biotin treatment to chase ManII-SBP-GFP along the ER-to-Golgi pathway (see Materials and methods). Golgi fractions were plotted in ( f ). ( g,h ) The Golgi export of TNFα does not require Arl15. In ( g ), after lentivirus-transduced knockdown of Arl15, HeLa cells transiently expressing RUSH reporter TNFα-SBP-GFP were incubated at 20 °C in the presence of biotin to accumulate TNFα-SBP-GFP at the Golgi. Cells were subsequently incubated at 37 °C to chase the reporter to exit the Golgi. The Golgi fractions were plotted in ( h ). Mon2 knockdown (Mon2 siRNA#2) serves as a control as it can accelerate the ER-to-Golgi transport and delay the Golgi export of secretory cargos. Scale bar, 10 µm. Error bar, mean ± SEM of n≥30 cells. ( i ) Arl15 is not required for TGFβ1-stimulated phosphorylation of Smad2/3. After lentivirus-transduced knockdown of Arl15, HeLa cells were serum-starved for 4 h followed by 20 hr treatment with starvation medium supplemented with or without 5 ng/ml TGFβ1. Cell lysates were subsequently immunoblotted for indicated proteins. ( j ) Depletion of Arl15 counteracts TGFβ1-induced transcriptions – it downregulates the transcription of N-cadherin, ID1, Snail1, vimentin, p27 kip1 , and p21 cip1 , and upregulates the transcription of E-cadherin and c-Myc. After lentivirus-transduced knockdown of Arl15, MDA-MB-231 cells were subjected to 16 hr starvation followed by 5 ng/ml TGFβ1 treatment for 4 hr. Transcripts of indicated genes were quantified and normalized as in . ( k ) Depletion of Arl15 upregulates the expression of E-cadherin in MDA-MB-231 cells. Knockdown was conducted as in ( j ). Cell lysates were immunoblotted for E-cadherin and GAPDH. In ( c , i , and k ) molecular weights (in kDa) are labeled in immunoblots. ( l ) Arl15-AL inhibits TGFβ1-induced transcription of SBE ×4 luc. The experiment was conducted as described in , except that all cells were treated with 5 ng/ml TGFβ1. In ( a , b , d , j , and l ) error bar, mean ± SD of n=3 experiments. p values were from the t -test (unpaired and two-tailed). NS, not significant (p>0.05); *, p≤0.05; **, p≤0.005; ***, p≤0.0005; ****, p≤0.00005; *****, p≤0.000005. Red dot, individual data point. Figure 6—figure supplement 1—source data 1. Uncropped gel and blot images for . The organization of the figure is similar to that of . Figure 6—figure supplement 1—source data 2. Numerical data for graphs in .

Article Snippet: The following antibodies were from Santa Cruz: mouse anti-Smad4 mAb (B-8, #sc-7966,1:1000 for WB), rabbit anti-Smad1/5/8 polyclonal antibody (pAb) (#sc-6031-R, 1:1000 for WB), rabbit anti-GAPDH pAb (#sc-25778, 1:1000 for WB), mouse anti-GFP mAb (#sc-9996, 1:1000 for WB, 1: 200 for IF), mouse anti-His mAb (#sc-8036, 1:1000 for WB), mouse anti-Myc mAb (#sc-40, 1:1000 for WB, 1:200 for IF) and mouse anti-HA mAb (#sc-7396, 1:1000 for WB).

Techniques: Activity Assay, Expressing, Luciferase, Plasmid Preparation, Control, Over Expression, Mutagenesis, shRNA, Knockdown, Incubation, Phospho-proteomics, Labeling, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Non-Canonical Targets of HIF1a Drive Cell-Type-Specific Dysfunction

doi: 10.1101/2020.04.03.003632

Figure Lengend Snippet: (A) Primary bioactives library screen showing the effect of 1753 molecules on percentage of oligodendrocytes (MBP+ cells/ total DAPI) formed by sgVhl OPCs relative to DMSO treated sgVhl OPCs. Anything above the dotted line represents a greater than 3-fold change increase in percentage of oligodendrocytes from DMSO (green dots). MEK inhibitors are highlighted as gray boxes with their respective drug names. (B) Pie charts of the number of non-toxic MEK inhibitors (in dark gray) compared to other classes of drugs (in blue) within top compound hits compared to their prevalence in the non-toxic compounds of the Selleck library. p-value was calculated using hypergeometric analysis. (C) Representative ICC images of oligodendrocytes (MBP+ in green) from the primary drug screen of the 5 top MEK inhibitor hits along with the DMSO negative control. Nuclei are marked by DAPI (in blue). Scale bars, 100µm. (D) Heatmap representation of the top 14 hits in an 8-point dose curve in 2-fold dilutions from 10µM to 78nM showing the fold change in the percentage (MBP+/total DAPI) of oligodendrocytes relative to DMSO treated sgVhl OPCs. The heatmap is shown as row Z-score, and rows are sorted by unsupervised hierarchical clustering with columns in order from high (10µM) to low dose (78nM). MEK inhibitors are highlighted in gray and bolded. Data are presented as the mean for each drug at each dose from 3 separate dose curve plates. (E) Collapsing all tested doses into one overall average shows the ability of each MEK inhibitor to increase the formation of oligodendrocytes (MBP+/DAPI) relative to DMSO treated sgVhl OPCs. AZD8330 and PD0325901 are shown in green representing the most effective drugs, while PD318088 and Selumetinib are shown in blue as slightly less effective drugs. Data are presented as the mean ± SD of the averages of all 8 doses for each drug from 3 separate dose curve plates. p-values were calculated using a one-way ANOVA with Tukey’s multiple comparisons test. (F) Representative Western blot for phosphorylated ERK1/2 (p-ERK1/2) relative to total ERK1/2 from whole cell lysates of sgVhl OPCs incubated with 100nM of AZD8330, PD0325901, PD318088 or Selumetinib for 30 minutes. Molecular weight is indicated to the right of the blot. (G) Quantification of the ratio of phosphorylated ERK1/2 relative to total ERK1/2 for AZD8330 and PD0325901 (both in green) and PD318088 and Selumetinib (both in blue). Data are presented as mean ± SD from 3 biological replicates (independent experiments) with a single technical replicate per experiment. p-values were calculated using one-way ANOVA with Dunnett’s multiple comparisons test. See also .

Article Snippet: A 3mM stock of the Selleck bioactive library in dimethylsulfoxide (DMSO) was then added to the plates using a 50nL solid pin tool attached to a Janus automated workstation (Perkin Elmer) at a 1:1000 dilution such that each well received a single compound at a final concentration of 3μM.

Techniques: Negative Control, Western Blot, Incubation, Molecular Weight

Journal: bioRxiv

Article Title: Non-Canonical Targets of HIF1a Drive Cell-Type-Specific Dysfunction

doi: 10.1101/2020.04.03.003632

Figure Lengend Snippet: Inhibition of MEK/ERK Signaling Increases Oligodendrocyte Formation from sgVhl OPCs, Related to . (A) Schematic depicting the procedure for the primary bioactives screen to uncover compounds that increase oligodendrocyte formation from sgVhl OPCs. (B) Primary bioactives library screen showing the effect of 1753 small molecules on number of oligodendrocytes (MBP+ cells) formed by sgVhl OPCs relative to DMSO treated sgVhl OPCs. Anything higher than the dotted line represents a greater than 3-fold change increase from DMSO (green dots). MEK inhibitors are highlighted as gray boxes with their respective drug names. (C) Primary screen positive control (Cas9 control+DMSO) and negative control (sgVhl+DMSO) percent oligodendrocyte (MBP+/DAPI) metrics on a per plate basis. Data represent mean ± SD from 16 technical replicates (individual wells) per plate per condition. (D) Representative immunocytochemistry images of oligodendrocytes (MBP+ in green) from primary screen positive (Cas9 control+DMSO) and negative (sgVhl+DMSO) controls. Nuclei are marked by DAPI (in blue). Scale bars, 100µm. (E) Schematic detailing the filtering steps starting with the bioactives library and narrowing down to top hits that are non-toxic (total DAPI FC<0.7), increased the number and percentage of oligodendrocytes (FC>3) and passed visual inspection to give the top 14 compound hits. Numbers in parentheses represent the number of drugs after each filtering step. (F) Quantification of the effect of all non-toxic MEK inhibitors (n=12) compared to all other non-toxic drugs from the primary screen (n=1472) on the percentage of oligodendrocytes from sgVhl OPCs relative to DMSO treated sgVhl OPCs. Data are presented as mean ± SD. p-values were calculated using the Mann-Whitney t-test. (G) Collapsing all tested doses into one overall average shows the ability of each MEK inhibitor to increase the formation of oligodendrocytes (MBP+/DAPI) from sgVhl.2 OPCs relative to DMSO treatment. AZD8330 and PD0325901 are shown in green representing the most effective drugs, while PD318088 and Selumetinib are shown in blue as slightly less effective drugs. Data are presented as the mean ± SD of all 8 doses for each drug from a single dose curve plate. (H) Table of IC50 values for MEK1 and MEK2 for the top MEK inhibitors AZD8330, PD035901, PD184352, and Selumetinib. IC50 is still currently unknown for PD318088. (I) List of the 12 drugs included on the MEK targets dose curve plate including the drug name and canonical target of the drug. (J) Heatmap representation of the 8-point dose curve performed for all 12 drugs in the MEK target dose curve plate shown as row Z-score. Data in the heatmap is the fold change in percent oligodendrocytes (MBP+/total DAPI) in sgVhl and sgVhl.2 OPCs relative to their respective DMSO negative controls. Rows were sorted by unsupervised hierarchical clustering and columns are in order from high (10µM) to low dose (78nM) of drug. ERK1/2 inhibitors are highlighted in gray and bold. Data are presented as the mean for each drug at each dose from 2 separate dose curve plates for both sgVhl and sgVhl.2 OPCs.

Article Snippet: A 3mM stock of the Selleck bioactive library in dimethylsulfoxide (DMSO) was then added to the plates using a 50nL solid pin tool attached to a Janus automated workstation (Perkin Elmer) at a 1:1000 dilution such that each well received a single compound at a final concentration of 3μM.

Techniques: Inhibition, Positive Control, Control, Negative Control, Immunocytochemistry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Non-Canonical Targets of HIF1a Drive Cell-Type-Specific Dysfunction

doi: 10.1101/2020.04.03.003632

Figure Lengend Snippet: (A) Western blot of nuclear lysates for HIF1a in sgVhl OPCs with 24hrs of 300nM AZD8330 treatment compared to DMSO with B-Actin as a loading control. Molecular weight is indicated to the right of the blot. (B) Violin plot of normalized number of transcripts (TPM values normalized to Cas9 control+DMSO) in Cas9 control+DMSO (in blue), sgVhl + DMSO (in green), and sgVhl + AZD8330 (in purple) relative to Cas9 control + DMSO of genes that were previously shown to be direct targets of HIF and increase in sgVhl OPCs compared to control (see ). p-values were calculated using the Kruskal Wallis One-Way ANOVA with Dunn’s multiple comparisons test. (C) Quantification of the normalized number of transcripts (TPM) for both Ascl2 and Dlx3 in Cas9 control+DMSO (in blue), sgVhl+DMSO OPCs (in green) and sgVhl+AZD8330 OPCs (in purple). OPCs were treated with either DMSO or 300nM AZD8330 for 14 hours. Data represent mean ± SD from 3 biological replicates (independent samples) from RNA-seq. (D) Gene set enrichment analysis (GSEA) analysis of gene program changes in sgVhl compared to Cas9 control OPCs demonstrates a significant reduction in GO terms for Oligodendrocyte Development (normalized enrichment score/NES = −2.74, FDR<0.0001, FWER p-val = 0.059) and Oligodendrocyte Differentiation (NES = −3.11, FDR<0.0001, FWER p-val = 0.002). (E) GSEA analysis of gene program changes in sgVhl + AZD8330 compared to sgVhl + DMSO OPCs demonstrates a significant enrichment in GO terms for Oligodendrocyte Development (normalized enrichment score/NES = 2.98, FDR= 5.5×10 −6 , FWER p-val = 0.004) and Oligodendrocyte Differentiation (NES = 3.58, FDR<1×10 −6 , FWER p-val <0.001). (F) Violin plot showing expression of transcripts (TPM values normalized to Cas9 control + DMSO OPCs) associated with GO term Oligodendrocyte Development (GO:0014003) that decrease (FC<0.75) in sgVhl + DMSO OPCs (in green) relative to Cas9 control + DMSO OPCs (in blue) as well as sgVhl OPCs following treatment with 300nM AZD8330. p-values were calculated using the Kruskal Wallis One-Way ANOVA with Dunn’s multiple comparisons test. (G) qRT-PCR of Sox10 and Myrf in Cas9 control+DMSO (in blue), sgVhl+DMSO (in green) and sgVhl+AZD8330 OPCs (in purple) normalized to endogenous control Rpl13a . OPCs were treated with either DMSO or 300nM AZD8330 for 14 hours. Data are presented as mean ± SEM from 4 technical replicates (individual wells). (H) Schematic of human brain oligocortical spheroids treated at days in vitro 70 with either DMSO or 300nM AZD8330 for 4 days. The spheroids were then cultured without drug until day 90 when they were incubated with hypoxyprobe, fixed and then sectioned for immunohistochemistry. (I) Representative immunohistochemistry images of DIV 90 oligocortical spheroids that had been treated from DIV 70-74 with either DMSO or 300nM AZD8330 for oligodendrocytes (MYRF+ in red) and hypoxic regions (hypoxyprobe in green). Scale bar, 100µM. (J) Quantification of oligodendrocytes (MYRF+ / mm 2 ) in the whole oligocortical spheroid (total), hypoxic region of the spheroid (hypoxyprobe+), and normoxic region of the spheroid (hypoxyprobe-) in DIV 90 spheroids that had been treated with either DMSO or 300nM AZD8330 from DIV 70-74. Data represent mean ± SD from 3 biological replicates (individual spheroids). p-values were calculated using Student’s two-tailed t-test. See also .

Article Snippet: A 3mM stock of the Selleck bioactive library in dimethylsulfoxide (DMSO) was then added to the plates using a 50nL solid pin tool attached to a Janus automated workstation (Perkin Elmer) at a 1:1000 dilution such that each well received a single compound at a final concentration of 3μM.

Techniques: Western Blot, Control, Molecular Weight, RNA Sequencing, Expressing, Quantitative RT-PCR, In Vitro, Cell Culture, Incubation, Immunohistochemistry, Two Tailed Test

Journal: bioRxiv

Article Title: Non-Canonical Targets of HIF1a Drive Cell-Type-Specific Dysfunction

doi: 10.1101/2020.04.03.003632

Figure Lengend Snippet: Inhibition of MEK/ERK Signaling Drives Sox10 Expression in sgVhl OPCs, Related to . (A) Schematic highlighting the setup of the RNA-seq experiment in which Cas9 control and sgVhl OPCs were treated with either DMSO or 300nM of AZD8330 for 14 hours and then cells were lysed for poly-adenylated mRNA extraction and sequenced. (B) qRT-PCR of Sox10 in sgVhl OPCs treated with DMSO, AZD0364 (1µM, in purple), SCH772984 (1µM, in magenta) or AZD8330 (300nM, in gray) for 14hrs normalized to endogenous control Rpl13a . Data are presented as mean ± SEM from 4 technical replicates (individual wells) per condition.

Article Snippet: A 3mM stock of the Selleck bioactive library in dimethylsulfoxide (DMSO) was then added to the plates using a 50nL solid pin tool attached to a Janus automated workstation (Perkin Elmer) at a 1:1000 dilution such that each well received a single compound at a final concentration of 3μM.

Techniques: Inhibition, Expressing, RNA Sequencing, Control, Extraction, Quantitative RT-PCR