Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: (A) Schematic of full length MAPKAP1 isoform 1 wild-type (WT) and the MAPKAP1 deletion (Del) proteins with domains highlighted. CRIM, Conserved Region In The Middle. RBD, Ras Binding Domain. PH, Pleckstrin Homology. (B) Microscale thermophoresis of labeled FHH:MAPKAP1RBD (16.8nM) with a titration series of GDP or GTPγs-loaded Ras. The binding curve is positive as the MST signal of the complex is higher than MAPKAP1RBD alone. MST-on time of 5s, n = 3 independent replicates. (C) BioID-western blot streptavidin pulldowns and input levels for birA* control, NRASWT, NRASQ61K and NRASQ61K with FHH:eGFP, MAPKAP1WT:FHH or MAPKAP1Del:FHH expression in CHL-1 cells. PI3K p110α subunit, Raf-1, and HA protein pulldown are controls. Pulldown normalized signal relative to control birA*:NRASQ61K shown below. (D) Quantification of mTOR and Rictor protein levels in the streptavidin pulldowns normalized to HA pulldown. All values and statistical tests relative to birA*:NRASQ61K, n=6 (Welch’s two-sided t-test). (E) PLA with endogenous Pan-Ras and mTOR or Rictor in MT NRAS MM485 melanoma cells. Scale bar, 20 μm. (F) PLA quantification in (E). n=3 independent experiments, 6-8 fields analyzed per condition per experiment (unpaired two-sided t-test). EV, empty vector. (G) Quantification of LocaTOR2 experiments with FHH:eGFP, MAPKAP1WT:FHH or MAPKAP1Del:FHH expression. All values relative to average of FHH:eGFP and MAPKAP1WT:FHH fold induction; n=3 (unpaired two-sided t-test). (H) Quantification of FHH:eGFP, MAPKAP1WT:FHH and MAPKAP1Del:FHH expression relative to MAPKAP1WT:FHH for all experiments graphed in (G). **p< 0.01, ***p< 0.001, ****p< 0.0001, and ns= not significant; all bar graphed data mean ± SEM. MST data are mean ± SD. See also Figure S6 and Table S4.

Article Snippet: pLEX-FHH-MAPKAP1 RBD /Triple Mutant (AA279-353; K307A,K310A,R312L) , This paper , Addgene Cat# 120711.

Techniques: Binding Assay, Microscale Thermophoresis, Labeling, Titration, Western Blot, Expressing, Plasmid Preparation

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: (A) Gene set enrichment analysis (GSEA) of RICTOR (Normalized enrichment score = −1.20) and RPTOR (NES= −1.30) knockdown signatures from primary human melanocytes comparing NRASWT versus NRASMT melanoma patient sample TCGA RNA expression data. *p< 0.05. (B) Western blot of protein remaining in control, RPTOR, RICTOR, MAPKAP1, and NRAS knockdown RNA-sequencing samples in MT NRAS melanoma cell line MM485. Labeled with normalized protein remaining relative to the control average. (C) Heat map showing log2(fold change) in RNA-sequencing of MT NRAS melanoma line, MM485, with RICTOR, MAPKAP1, RPTOR or NRAS knockdown compared to control. mTORC2 gene signature is derived from concordant genes between shRICTOR and shMAPKAP1 samples and mTORC1 signature between shRPTOR samples. (D) Heatmap of −log10(adjusted p-values) for top GO terms for NRAS-regulated shMTORC1 and shMTORC2 down regulated gene sets as labeled in (C). Unfiltered shNRAS down regulated gene set analysis for comparison. (E) Heatmap of −log10(FDR q-values) for GSEA positively enriched REACTOME or KEGG or (F) Pathway interaction database (PID) signatures from Molecular Signatures Database with MTOR, RICTOR, MAPKAP1 or RPTOR expression as the phenotype in MT NRAS TCGA melanoma patient samples. Relevant signatures (red). See also Figure S7, Tables S5 and S6.

Article Snippet: pLEX-FHH-MAPKAP1 RBD /Triple Mutant (AA279-353; K307A,K310A,R312L) , This paper , Addgene Cat# 120711.

Techniques: RNA Expression, Western Blot, RNA Sequencing Assay, Labeling, Derivative Assay, Expressing

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: (A) BioID-western blot showing mTOR protein levels in the streptavidin pulldowns of birA*:NRASQ61K with shGFP, shRPTOR, shRICTOR or shMAPKAP1 with two independent hairpins in CHL-1 cells. PI3K p110α subunit, Raf1, and HA protein levels in streptavidin pulldowns are controls. Pulldown and input normalized values relative to the control shown below. (B) Quantification of mTOR, p110α and Raf1 protein levels in the streptavidin pulldown Normalized to HA pulldown and respective input levels and relative to shGFP mean, n=5 (unpaired two-sided t-test). (C) Quantification of protein remaining after knockdown compared to the average of controls for (B). (D) PLA in MT NRAS MM415 melanoma cells with endogenous Ras and mTOR with control, mTORC1 or mTORC2 component knockdown. Scale bar, 20 μm. (E) Quantification of PLA shown in (D). n=2 independent hairpins per knockdown. Relative to the mean of control knockdowns. (unpaired two-sided t-test). (F) Quantification of protein remaining after knockdown relative to mean signal of control knockdowns for PLA in (E). (G) Western blot of HA co-immunoprecipitation of empty vector (EV), FHH:NRASWT or FHH:NRASQ61K with endogenous Rictor, MAPKAP1 and Raptor in wild-type Ras CHL-1 cells. (H) Quantification of HA co-IP experiments as in (G). Values normalized to HA pulldown signal and relative to NRASWT signal. n= 5 or 8 (Welch’s two-sided t-test). (I) Western blot showing mTOR protein levels in the input and streptavidin pulldowns of birA* control, NRASWT, NRASQ61K and NRASQ61K Effector Domain Alanine point mutants. (*) non-specific background band. (J) Quantification of streptavidin pulldown protein levels as in (I). mTOR signal normalized to HA signal. All values relative to birA*: NRASQ61K, n=3 (Welch’s two-sided t-test relative to Q61K). All graphed data are mean ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001, ns= not significant. See also Figure S5.

Article Snippet: pLEX-FHH-MAPKAP1 RBD /Triple Mutant (AA279-353; K307A,K310A,R312L) , This paper , Addgene Cat# 120711.

Techniques: Western Blot, Immunoprecipitation, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: (A) Western blot of replicate in vitro mTORC2 IP-kinase assays with a GST-Akt1 tail substrate. Input levels right panel. HA-tagged MAPKAP1 pulldown of mTORC2 from 293T cells co-expressing empty vector (EV) or FLAG-GTPase. 5 μM PP242 (mTORi). (B) Quantification of the GST normalized mTORC2 IP-kinase assay pAktS473 signal, as in (A), relative to mean of the untreated EV control. n= 4-6 (unpaired two-sided t-test). (C) Quantification of HA IP mTORC2 kinase assay with addition of indicated recombinant GDP-loaded WT or GTPγS-loaded G12V H-Ras in vitro relative to EV in buffer only. n=5-6; ** p < 0.01, (unpaired two-sided t-test). (D) Diagram of the LocaTOR2 assay for subcellular compartment-specific in vivo mTORC2 kinase activity. (E) LocaTOR2-based subcellular compartment mTORC2 activity quantified as phosphorylated Frb:Akt2S474 signal over untreated in MT NRAS MM485 melanoma cells. 500 nM KU-0063794 (Pan-mTORi). Dotted line at 2. n=2 experimental replicates. (F) Quantification of compartment-specific LocaTOR2 signal with shSCR or shNRAS knockdown, n=3. (Welch’s two-sided t-test to respective shSCR). (G) Quantification of protein remaining after knockdown compared to shSCR for (F). All data are mean ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001. See also Figure S6.

Article Snippet: pLEX-FHH-MAPKAP1 RBD /Triple Mutant (AA279-353; K307A,K310A,R312L) , This paper , Addgene Cat# 120711.

Techniques: Western Blot, In Vitro, Expressing, Plasmid Preparation, IP-Kinase Assay, Kinase Assay, Recombinant, In Vivo, Activity Assay

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pLEX-FHH-MAPKAP1 RBD /Triple Mutant (AA279-353; K307A,K310A,R312L) , This paper , Addgene Cat# 120711.

Techniques: Western Blot, Transduction, In Situ, Recombinant, Lysis, Protease Inhibitor, Staining, Magnetic Beads, Clone Assay, Bicinchoninic Acid Protein Assay, Isolation, Labeling, Viability Assay, RNA Sequencing Assay, Sequencing, Expressing, CRISPR, Mass Spectrometry, Mutagenesis, shRNA, Plasmid Preparation, Luciferase, Software, Blocking Assay

Journal: Nature Communications

Article Title: Clearance of maternal barriers by paternal miR159 to initiate endosperm nuclear division in Arabidopsis

doi: 10.1038/s41467-018-07429-x

Figure Lengend Snippet: Both maternal and paternal miR159 are required for seed development. a Representative F1 siliques from 6 DAP (Day After Pollination) ♀Col-0 × ♂Col-0 and ♀Col-0 × ♂ mir159abc , respectively. Smaller one from three lower panels indicates aborted seeds when Col-0 was pollinated by pollen of the mir159abc triple mutant. F1 siliques were divided into 4 categories according to % of seed set per silique: >90% (+), 60–90% (++), 30–60% (+++), <30% (++++), respectively. Source data are provided in the accompanying Source Data file. b Statistical analysis of seed set from a . The percentage of seed set per silique for each genotype is shown. One hundred siliques for each genotype were shown, and each dot represents one silique. c Statistical analysis of average perimeter per seed from each category. Perimeters of 20 seeds randomly picked from indicated genotypes were measured using the Image J software. Box plot shows the distribution of each seed perimeter for each genotype, and asterisks indicate a significant difference between the indicated samples ( t -tests, P -value <0.05). d The release and migration of twin sperm in the developing seeds of Col-0 and the mir159abc triple mutant. Pistils from Col-0 and the mir159abc triple mutant harboring the HTR10-GFP transgene were self-pollinated, and the developing seeds at indicated time were dissected and imaged by fluorescence microscope. >50 developing seeds for each genotype were analyzed and representative images were shown. The yellow arrows indicate two released sperm. Embryo sacs are green due to auto-fluorescence. Scale bar, 20 µm

Article Snippet: The mir159abc triple mutant was kindly provided by Dr. Anthony A. Millar, which was constructed by three independent T-DNA insertional mutants (SAIL_430_F11 for mir159a ; SAIL_770_GO5 for mir159b ; and SAIL_248_G11 for mir159c ) .

Techniques: Mutagenesis, Software, Migration, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Clearance of maternal barriers by paternal miR159 to initiate endosperm nuclear division in Arabidopsis

doi: 10.1038/s41467-018-07429-x

Figure Lengend Snippet: Lack of paternal miR159 caused defective endosperm nuclear divisions. a – f Endosperm nuclear divisions of the developing seeds from hand-pollinated Col-0 ( a , c , e ) or the F1 progenies of ♀Col-0 × ♂ mir159abc ( b , d , f ) at indicated time (HAP, hour after pollination) by DIC imaging. White stars indicate endosperm nuclei or the central cell nucleus. Yellow stars indicate the fused nucleus by the egg cell and the sperm. Yellow dashed lines indicate the developing embryo. g Statistical analysis of endosperm nuclei number at 24 HAP. Seeds from hand-pollinated ♀Col-0 × ♂Col-0, ♀ mir159abc × ♂Col-0, ♀Col-0 × ♂ mir159abc , ♀ mir159abc × ♂ mir159abc , and ♀Col-0 × ♂ mir159ab , respectively, were observed by DIC imaging and >200 F1 seeds from three biological replicates were examined for each category. Biological replicates for each genotype were shown by dots. h proFWA::RFP reporter in the unfertilized ovule (the top panels), as the female pollinated with Col-0 (the middle panels) or mir159abc (the bottom panels) at indicated time (HAP) by fluorescence microscopy. White arrows indicate RFP signals. Auto-fluorescence of the embryo sac is shown in the top middle panel. % indicates the ratio of developing seeds similar to the representative image relative to total ones. n represents total numbers of analyzed developing seeds for each category. Scale bar, 20 µm

Article Snippet: The mir159abc triple mutant was kindly provided by Dr. Anthony A. Millar, which was constructed by three independent T-DNA insertional mutants (SAIL_430_F11 for mir159a ; SAIL_770_GO5 for mir159b ; and SAIL_248_G11 for mir159c ) .

Techniques: Imaging, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Clearance of maternal barriers by paternal miR159 to initiate endosperm nuclear division in Arabidopsis

doi: 10.1038/s41467-018-07429-x

Figure Lengend Snippet: Subcellular localization of proMYB33::MYB33-GFP in the developing seeds. a , b Fluorescence images of the proMYB33::MYB33-GFP/proFWA::RFP doubly transgenic plants in the unfertilized ovule a and the developing seed at 12 HAP b . White dashed circles indicate the RFP signal, and the white arrowheads indicate the GFP signal. proFWA::RFP was used as the marker for the central cell/endosperm nuclei. Embryo sacs are green or red due to auto-fluorescence. c – e Fluorescence images of the developing seeds from the proMYB33::MYB33-GFP transgenic plants pollinated with pollen of Col-0 at 24 h c , mir159abc at 12 HAP d and 24 HAP e , respectively. All the left panels show the auto-fluorescence control of the developing seeds under the RFP channel, and the overlay was performed for each. White arrowheads indicate the GFP signal. % indicates the ratio of those developing seeds similar to the representative image relative to total ones. n represents total numbers of analyzed embryo sacs. Scale bar, 20 µm. f Quantification of proMYB33::MYB33-GFP signals from a – e . “unfertilized” indicates mature ovules of the proMYB33::MYB33-GFP transgenic plants as shown in the middle panel from a . Other columns represent that proMYB33::MYB33-GFP transgenic plants were pollinated as indicated time (12 and 24 HAP) by pollen of Col-0 or mir159abc , respectively. An arrow flanking each endosperm nucleus was used for the quantification of GFP signal intensity as shown in Supplementary Fig. , and the measurements were performed using the Image-Pro Insight software. The GFP signal intensity was calculated by the number measured from the endosperm nucleus relative to the average number of flanking regions along the arrow. One small circle indicates the result from one image. Error bars show SD calculated from ~10 developing seeds for each category, and asterisks indicate a significant difference between the indicated samples ( t -tests, P -value <0.05). n.s. indicates non-significant

Article Snippet: The mir159abc triple mutant was kindly provided by Dr. Anthony A. Millar, which was constructed by three independent T-DNA insertional mutants (SAIL_430_F11 for mir159a ; SAIL_770_GO5 for mir159b ; and SAIL_248_G11 for mir159c ) .

Techniques: Fluorescence, Transgenic Assay, Marker, Control, Software

Journal: Nature Communications

Article Title: Clearance of maternal barriers by paternal miR159 to initiate endosperm nuclear division in Arabidopsis

doi: 10.1038/s41467-018-07429-x

Figure Lengend Snippet: Detection of 3′ cleavage products of MYB33 and MYB65 by 5′RACE-PCR. a Alignment of miR159 and its targeting sites on MYB33 and MYB65 . Mutated nucleotides of miR159 binding sites are highlighted in red as mMYB33 . Vertical lines and dots indicate complementary nucleotides and uncomplimentary one, respectively. The arrows indicate the cleavage site of miR159 on the coding region of MYB33 or MYB65 . a Schematics of MYB33 and MYB65 coding regions, the location of miR159 binding sites, positions of primers used in c – d . Red rectangles indicate miR159 targeting sites. Arrows indicate the orientation of primers. c , d Semi-quantitative 5′RACE-PCR to detect 3′ cleavage fragments generated through miR159-guided cleavage of MYB33 and MYB65 transcripts. UBQ5 is a loading control. M indicates the DNA ladder, and standard sizes were labeled. For c , we used pistils from Col-0, and pollinated it using pollen from Col-0 or the mir159abc triple mutant. For d , we used pistils from the proFWA::mMYB33-GFP transgenic plants, and pollinated it using pollen from Col-0. The experiments were performed with two biological replicates. Source data are provided in the accompanying Source Data file

Article Snippet: The mir159abc triple mutant was kindly provided by Dr. Anthony A. Millar, which was constructed by three independent T-DNA insertional mutants (SAIL_430_F11 for mir159a ; SAIL_770_GO5 for mir159b ; and SAIL_248_G11 for mir159c ) .

Techniques: Binding Assay, Generated, Control, Labeling, Mutagenesis, Transgenic Assay