Journal: bioRxiv

Article Title: Identification of Gip as a novel phage-encoded gyrase inhibitor protein featuring a broad activity profile

doi: 10.1101/2021.01.28.428610

Figure Lengend Snippet: Cultivation of a wildtypic SOS reporter strain ATCC 13032::P recA - venus , a wiltypic prophage reporter strain ATCC 13032::P lys - eyfp and an SOS-deficient prophage reporter strain ATCC 13032 Δ recA ::P lys - eyfp carrying the plasmids pAN6 or pAN6-cg1978 was performed in the BioLector ® microcultivation system in CGXII-Kan 25 medium with 2 % (w/v) glucose and 50 μM IPTG. All data represent mean values with standard deviations from three independent biological triplicates (n=3). (A) Growth curves based on the backscatter measurements in the BioLector ® microcultivation system. The elongated lag-phase of the Cg1978 overproducing strain is marked in grey. (B) Percentage of induced cells based on the flow cytometric measurements of eYFP or Venus fluorescence of the reporter strains. (C) Time-lapse fluorescence microscopy of the C. glutamicum prophage reporter strain ATCC 13032::P lys - eyfp carrying the pAN6-cg1978 plasmid. Cells were grown in PDMS-based microfluidic chip devices using CGXII medium supplemented with 25 μg ml −1 kanamycin. The medium was continuously supplied with a flow rate of 300 nl min −1 . Overexpression of cg1978 was induced by addition of 50 μM IPTG (Video S1 and S2).

Article Snippet: Besides the wildtypic prophage reporter strain ATCC 13032::P lys - eyfp , a wildtypic SOS reporter strain ATCC 13032::P recA - venus as well as an SOS-deficient prophage reporter strain ATCC 13032 Δ recA ::P lys - eyfp lacking the co-protease RecA – required for the induction of the host SOS response ( ) – were used.

Techniques: Fluorescence, Microscopy, Plasmid Preparation, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: Icaritin promotes the osteogenesis of bone marrow mesenchymal stem cells via the regulation of sclerostin expression

doi: 10.3892/ijmm.2020.4470

Figure Lengend Snippet: SOST overexpression reverses icaritin-induced osteogenesis of hBMSCs. (A) Western blot analysis for the protein level of SOST. (B) The mRNA expression of SOST in BMSCs, BMSCs-vector, and BMSCs-SOST. (C) Mineralization in cultured hBMSCs in BMSCs-vector and BMSCs-SOST groups with or without icaritin were detected at day 14. Magnification, ×10. (D) The mRNA levels of OCN, Runx2, Alp and β-actin were determined by reverse transcription quantitative polymerase chain reaction. Data are presented as mean ± standard deviation (n=3). ** P<0.01 vs. BMSCs group; # P<0.05 and ## P<0.01 vs. BMSCs-vector group. SOST, sclerostin; BMSCs, bone marrow-derived mesenchymal stem cells; OCN, osteocalcin; Runx2, RUNX family transcription factor 2; Alp, alkaline phosphatase.

Article Snippet: Following fixing with 4% paraformaldehyde at 4°C for 15 min, the hBMSCs were treated with 0.25% Triton X-100 and 2% bovine serum albumin at 4°C for 15 and 30 min, respectively. hBMSCs were washed and incubated overnight with primary anti-SOST antibody (1:100; cat. no. MAB1406; R&D Systems, Inc.) at 4°C.

Techniques: Over Expression, Western Blot, Expressing, Plasmid Preparation, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Icaritin promotes the osteogenesis of bone marrow mesenchymal stem cells via the regulation of sclerostin expression

doi: 10.3892/ijmm.2020.4470

Figure Lengend Snippet: Icaritin downregulates SOST, which is reversed by ICI 182780. hBMSCs were cultured in osteogenic induction medium for 3, 7 and 14 days, and SOST protein levels were measured. DMSO was served as the control group. (A) SOST protein expression was measured by western blot analysis. (B) Band density of SOST was quantified by densitometry. Data are presented as mean ± standard deviation (n=3). ** P<0.01 vs. control group at the same day. SOST, sclerostin; BMSCs, bone marrow-derived mesenchymal stem cells; OCN, osteocalcin; Runx2, RUNX family transcription factor 2; Alp, alkaline phosphatase.

Article Snippet: Following fixing with 4% paraformaldehyde at 4°C for 15 min, the hBMSCs were treated with 0.25% Triton X-100 and 2% bovine serum albumin at 4°C for 15 and 30 min, respectively. hBMSCs were washed and incubated overnight with primary anti-SOST antibody (1:100; cat. no. MAB1406; R&D Systems, Inc.) at 4°C.

Techniques: Cell Culture, Control, Expressing, Western Blot, Standard Deviation, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Icaritin promotes the osteogenesis of bone marrow mesenchymal stem cells via the regulation of sclerostin expression

doi: 10.3892/ijmm.2020.4470

Figure Lengend Snippet: Effects of icaritin + ICI 182780 on the expression of sclerostin detected by immunofluorescence staining. hBMSCs were divided into three groups: Icaritin; icaritin + ICI 182780; and DMSO. Following culture in osteogenic induction medium for 14 days, all the groups were collected for immunofluorescence staining. Sclerostin was labeled with green fluorescence and the nuclei were stained with DAPI.

Article Snippet: Following fixing with 4% paraformaldehyde at 4°C for 15 min, the hBMSCs were treated with 0.25% Triton X-100 and 2% bovine serum albumin at 4°C for 15 and 30 min, respectively. hBMSCs were washed and incubated overnight with primary anti-SOST antibody (1:100; cat. no. MAB1406; R&D Systems, Inc.) at 4°C.

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Fluorescence

Journal: Journal of cellular biochemistry

Article Title: Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

doi: 10.1002/jcb.25432

Figure Lengend Snippet: Fig. 1. Sclerostin enhances adipocyte differentiation in 3T3-L1 cells. Confluent 3T3-L1 cells were incubated in differentiation media and treated with indicated doses of sclerostin at day 0 postinitiation of differentiation. Cells were fixed and lipid accumulation was monitored by oil red O staining at day 5 of differentiation (A). Lipid staining was extracted using isopropyl alcohol and oil red O accumulation quantified by measuring absorbance at 520 nm. Fold-increase in absorbance over nontreated cells is presented (B). After total RNA was extracted from the cells at day 5 of differentiation, adiponectin or peroxisome proliferator-activated receptor (PPAR) g mRNA level was determined by qRT- PCR (C). The lipoprotein lipase (LPL) and the fatty acid-binding protein 4 (Fabp4) mRNA levels were determined by qRT-PCR (D). b-actin was used as an endogenous control. Data are presented as means S.D.; n ¼ 3, , P < 0.05 versus absence of sclerostin (0).

Article Snippet: Recombinant mouse sclerostin and Wnt3a were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Staining, Quantitative RT-PCR, Binding Assay, Control

Journal: Journal of cellular biochemistry

Article Title: Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

doi: 10.1002/jcb.25432

Figure Lengend Snippet: Fig. 2. Sclerostin attenuates Wnt3a-inhibited adipocyte differentiation and expression of LRP5 and LRP6 in 3T3-L1 cells. (A and B) Confluent 3T3-L1 cells were incubated in differentiation media (DM þ) or none (DM ) and treated with indicated doses of sclerostin (20 ng/mL), Wnt3a (10 ng/mL), sclerostin (50 ng/mL) and Wnt3a (10 ng/mL), or vehicle (). At day 5, total RNA was extracted from the cells and adiponectin and PPARg mRNA levels were determined by qRT-PCR. GAPDH was used as an endogenous control. Data are presented as means S.D.; n ¼ 3, , P < 0.05. (B) Photograph of oil red O staining (left panel) and quantification of oil red O staining at day 5 (right panel). (C) LRP4, LRP5, and LRP6 mRNA expression were determined by RT-PCR in 3T3-L1 preadipocytes (A) or MC3T3-E1 osteoblasts (O). b-actin was used as a positive control. Lane M represents the size marker (100-bp ladder).

Article Snippet: Recombinant mouse sclerostin and Wnt3a were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Control, Staining, Reverse Transcription Polymerase Chain Reaction, Positive Control, Marker

Journal: Journal of cellular biochemistry

Article Title: Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

doi: 10.1002/jcb.25432

Figure Lengend Snippet: Fig. 3. Sclerostin regulates C/EBPb expression but not cell proliferation and or caspase-3/7 activity in 3T3-L1 cells. (A) Confluent 3T3-L1 cells were incubated in differentiation media and treated with sclerostin (20 ng/mL). After 24 h, the level of C/EBPb protein in the cells was determined by Western blot analysis. (B) Confluent 3T3-L1 cells were incubated in differentiation media and treated with sclerostin (20 ng/mL), and then cultured for further 24 h. DNA synthesis of 3T3-L1 cells was measured by BrdU incorporation using an ELISA kit. BrdU incorporation in the absence of sclerostin is adjusted to 1. (C) Confluent 3T3-L1 cells were incubated in differentiation media and treated with sclerostin (20 ng/mL). After 4 h, cellular caspase-3/7 activities were measured. Fold-increase in activity was calculated based on activity measured in control (absence) cells. Each assay represents a separate experiment performed in triplicate. Data are presented as means S.D; n ¼ 3; n.s. (no significant difference).

Article Snippet: Recombinant mouse sclerostin and Wnt3a were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Activity Assay, Incubation, Western Blot, Cell Culture, DNA Synthesis, BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of cellular biochemistry

Article Title: Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

doi: 10.1002/jcb.25432

Figure Lengend Snippet: Fig. 4. Effects of TAZ knockdown on sclerostin-mediated adipocyte differentiation in 3T3-L1 cells. (A) 3T3-L1 cells were incubated in differentiation media and then transiently cotransfected in 24-well plates with a TAZ reporter plasmid 8xGTIIC-Lux. Then cells were treated with sclerostin (20 ng/mL), Wnt3a (10 ng/mL), sclerostin (20 ng/mL), and Wnt3a (10 ng/mL) or vehicle () for 6 h, after which luciferase activity was determined. Normalized luciferase activity is shown as the ratio of luciferase activity relative to 8xGTIIC-Lux with vehicle, which is set to a value of 1. (B) Confluent 3T3-L1 cells were incubated in differentiation media and treated with sclerostin (20 ng/mL), Wnt3a (10 ng/ mL), sclerostin (50 ng/mL) and Wnt3a (10 ng/mL) or vehicle () for 5 days. Total RNA was extracted from the cells and then ctgf mRNA level was determined by qRT-PCR. (C, D, E, and F) 3T3-L1 cells were transiently transfected with TAZ siRNA (siTAZ) or control siRNA (siCont) (both at 10 nM) at day 0. Then cells were treated with sclerostin (20 ng/mL) or vehicle (). At day 2, the levels of TAZ protein in the cells were determined by Western blot analysis. (D) Photographs of oil red O staining and (E) quantification of oil red O staining in 3T3-L1 cells at day 6. Graph showing fold-increase in absorbance over nontreated cells. (F) A qRT-PCR was performed to quantify mRNA expression level of adiponectin. b-actin was used as an endogenous control. Data are presented as means S.D; n ¼ 3; , P < 0.05.

Article Snippet: Recombinant mouse sclerostin and Wnt3a were purchased from R&D Systems (Minneapolis, MN).

Techniques: Knockdown, Incubation, Plasmid Preparation, Luciferase, Activity Assay, Quantitative RT-PCR, Transfection, Control, Western Blot, Staining, Expressing

Journal: Journal of cellular biochemistry

Article Title: Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

doi: 10.1002/jcb.25432

Figure Lengend Snippet: Fig. 5. Effects of TAZ over-expression on sclerostin-mediated adipocyte differentiation in 3T3-L1 cells. 3T3-L1 cells were transiently transfected in 24-well plates with a TAZ expression plasmid pTAZ or empty vector pcDNA3 (both at 0.1 mg/well). After one day, cells were treated with sclerostin (20 ng/mL) or vehicle () (day 0). The levels of TAZ protein in the cells were determined by Western blot analysis at day 2 (A). Photographs of oil red O staining (B) and quantification of oil red O staining (C) in 3T3-L1 cells at day 6. Total RNA was extracted from the cells at day 6 and then a qRT-PCR was performed to quantify mRNA expression level of adiponectin (D). b-actin was used as an endogenous control. Data are presented as means S.D; n ¼ 3; n.s. (no significant difference).

Article Snippet: Recombinant mouse sclerostin and Wnt3a were purchased from R&D Systems (Minneapolis, MN).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Quantitative RT-PCR, Control

Journal: Nature methods

Article Title: Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer

doi: 10.1038/nmeth.2331

Figure Lengend Snippet: LVV integrations in HCCs at CIS targeted different genes: ( a ) Braf ( b ) Fign , ( c ) Sos1 , ( d ) Rtl1 within the Dlk1-Dio3 region. Dashed lines: intergenic chromosomal regions; solid lines and boxes: introns and exons of genes in the region, respectively. Grey triangles: transcript orientation; bended arrows: integration position and vector orientation. Below are represented the aberrant transcripts generated by LVV integration. V: vector-derived exon containing a portion of the LVV LTR and leader sequence up to the LVV splice donor; boxes: genomic exons; dashed lines: splicing events. ( e ) Representative functional domains of BRAF and SOS1 proteins and schematic of truncated proteins generated by LVV integration. Aminoacid number at the predicted truncation is indicated. ( f ) Vector design for the liver gene transfer of candidate cancer genes, based on LVV with SIN LTRs. ( g ) Liver tumor incidence in different experimental groups administered with SINLVs that express candidate cancer genes. Tr = truncated ORF; n = number of mice. ( h, i ) Liver of a 64-days-old Cdkn2 −/− Ifnar1 −/− mouse expressing truncated Fign ( h), and of a 349-days-old wild type mouse expressing Rtl1 and treated with CCl 4 (i).Arrows indicate 2 HCCs. Scale bar = 1 cm. ( j-l ) Representative hematoxylin and eosin stained sections of HCC from mice treated with the SINLV overexpressing full length Fign ( j ), truncated Fign ( k ) or truncated Sos1 ( l ). * indicates tumor area. Scale bar= 100 μm. See also and - .

Article Snippet: The Taqman gene expression assays that we used are: Hs00269944-m1 (for the detection of BRAF ); Hs00250679-s1 (for the detection of FIGN ); Hs00893134_m1 (for the detection of SOS1 ); Hs00194538-m1 (for the detection of SFRS4 ) (Applied Biosystems).

Techniques: Plasmid Preparation, Generated, Derivative Assay, Sequencing, Functional Assay, Expressing, Staining

Journal: Nature methods

Article Title: Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer

doi: 10.1038/nmeth.2331

Figure Lengend Snippet: ( a ) Each tumor bearing integrations targeting a CIS is represented as a square with color according to the grade.( b ) The heatmap shows expression levels of LVV induced HCCs and non-tumor liver from experimental mice. Magenta indicates low expression and turquoise indicates high expression. ( c ) Plotted is the fold change of expression (versus expression in normal livers) of HCCs bearing the indicated CIS integration. Each plot shows every probe of the microarray probeset for the CIS gene (from 5′ to 3′ of each transcript). The black triangle and dashed grey line indicate LVV integration landing inside the transcript ( Braf , Sos1 and Fign ).

Article Snippet: The Taqman gene expression assays that we used are: Hs00269944-m1 (for the detection of BRAF ); Hs00250679-s1 (for the detection of FIGN ); Hs00893134_m1 (for the detection of SOS1 ); Hs00194538-m1 (for the detection of SFRS4 ) (Applied Biosystems).

Techniques: Expressing, Microarray

Journal: Nature methods

Article Title: Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer

doi: 10.1038/nmeth.2331

Figure Lengend Snippet: ( a ) Heat map and dendrogram showing hierarchical unsupervised clustering analysis of LVV-induced HCCs and nontumoral livers from experimental mice. CIS genes hit in the HCCs are indicated above the heat map. No CISs are HCCs without integrations at CIS genes. Asterisks indicate HCCs with additional integrations at CISs. Expression levels in the heat maps are color coded from blue (low) to red (high). ( b ) Expression profile of Rtl1 HCCs as compared to pooled HCCs (three with Braf integration outlined in pink, six with Fign integration in green, two with Sos1 integration in violet and two without integration at CIS in yellow) and nontumoral livers from the different genetic backgrounds (N, in white) by GSEA. A heat map representation of the 15 most overexpressed genes of oxidative phosphorylation between Rtl1 HCCs and other samples is shown (from blue, low expression, to red, high expression). GSEA statistics: NES, normalized enrichment score; FDR q value: false discovery rate; FWER P value, family-wise error rate. Bottom, enrichment plot showing the overrepresentation at the top and bottom of the ranked gene set. ( c ) Expression profile of Fign HCCs as compared to eight nontumoral livers. Heat map representations of the most upregulated (top) and downregulated (bottom) genes of the WNT signaling pathway in Fign tumors versus expression in nontumoral livers are shown. Bottom, enrichment plot as in b. (See also , and .)

Article Snippet: The Taqman gene expression assays that we used are: Hs00269944-m1 (for the detection of BRAF ); Hs00250679-s1 (for the detection of FIGN ); Hs00893134_m1 (for the detection of SOS1 ); Hs00194538-m1 (for the detection of SFRS4 ) (Applied Biosystems).

Techniques: Expressing, Phospho-proteomics

Journal: Nature methods

Article Title: Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer

doi: 10.1038/nmeth.2331

Figure Lengend Snippet: ( a–c ) Expression fold changes for SOS1 (a), FIGN (b) and BRAF (c) for nontumoral liver (non-tum) and HCCs from the HSR and MSSM collections. The MSSM collection25 was analyzed by Affymetrix microarray, whereas the HSR collection was analyzed by RT-qPCR. Black lines, mean; colored whiskers, s.d. Black P value by unpaired t-test; green P value by two-tailed Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001 (see also ). ( d-f ) Comparative genomic hybridization data of the genomic regions encompassing FIGN, BRAF and RTL1 were obtained by consulting the OncoDB.HCC database . Graphs show a chromosomal region of ±10 megabase pairs centered at the gene of interest (arrow). Bars: pink, copy-number gain; red, minimal overlap region of copy-number gain; brown, amplification; pale green, copy-number loss; dark green, minimal overlap region of copy-number loss. ( g ) Survival curves for patients with HCC27 with high or low expression of SOS1 (Online Methods). GBW, Gehan-Breslow-Wilcoxon test; LR, log-rank Mantel-Cox test. ( h ) Clustering of human HCCs27 was performed considering the human orthologs of the upregulated (UP) genes from the murine Braf signature (Online Methods). Unsupervised clustering analysis identified two main HCC clusters (blue and red boxes). The yellow box marks genes highly expressed in the cluster with poorer prognosis and that mainly have IPA (Ingenuity Systems pathway analysis software) biological functions of Cell Cycle, DNA Replication and Cancer . Magenta, low expression; cyan, high expression. (i) Disease-free survival of the patients with HCC belonging to the clusters identified in h.

Article Snippet: The Taqman gene expression assays that we used are: Hs00269944-m1 (for the detection of BRAF ); Hs00250679-s1 (for the detection of FIGN ); Hs00893134_m1 (for the detection of SOS1 ); Hs00194538-m1 (for the detection of SFRS4 ) (Applied Biosystems).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Two Tailed Test, Hybridization, Amplification, Software

Journal: Scientific Reports

Article Title: RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

doi: 10.1038/s41598-018-24969-w

Figure Lengend Snippet: SOS1 is up-regulated in RUNX1 high-expressing gastric cancers. ( a , b ) Identification of genes under the control of RUNX1 transcription factor. Sixteen genes were commonly up-regulated in 5 independent gastric cancer patients with a higher RUNX1 expression (GSE62254, GSE29272, GSE35809, GSE34942 and GSE22377). ( c ) Up-regulation of SOS1 in the gastric cancer cell lines relative to the normal stomach tissue (total protein lysate of the whole stomach tissue: Catalog #: P1234248, Lot #: A208104, BioChain). Cell lysates were processed for immunoblotting with the indicated antibodies. ( d ) Down-regulation of SOS1 expression upon RUNX1 inhibition. Non-depleted and RUNX1 -depleted NUGC4 and MKN45 cells were incubated with 3 μM doxycycline. Twenty-four hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of control vector-transduced cells (n = 3). ( e ) Down-regulation of SOS1 expression upon RUNX1 inhibition in NUGC4 and MKN45 cells. Non-depleted and RUNX1 -depleted cells were treated as in ( d ). Forty-eight hours after treatment, cell lysates were prepared and subjected to immunoblotting. Data are mean ± SEM values. *P < 0.05, ***P < 0.001, by two-tailed Student’s t-test.

Article Snippet: Anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology).

Techniques: Expressing, Control, Western Blot, Inhibition, Incubation, Quantitative RT-PCR, Plasmid Preparation, Two Tailed Test

Journal: Scientific Reports

Article Title: RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

doi: 10.1038/s41598-018-24969-w

Figure Lengend Snippet: Indispensable role of SOS1 in the ErbB2/HER2 signaling cascade. ( a ) Efficacy of shRNAs targeting SOS1 . MKN45 cells were transduced with control (sh_ Luc ) or with SOS1 shRNAs (sh_ SOS1 #1 and #2) and cultured in the presence of 3 μM doxycycline. Twenty-four hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of control vector-transduced cells (n = 3). ( b ) SOS1 -depletion-mediated dephosphorylation of HER2. Non-depleted and SOS1 -depleted MKN45 cells were treated as in ( a ). Forty-eight hours after treatment, cell lysates were processed for immunoblotting. ( c ) Growth curves of MKN45 cells transduced with control (sh_ Luc ) or with SOS1 shRNAs (sh_ SOS1 #1 and #2) in the presence of 3 μM doxycycline (n = 5). ( d ) Efficacy of shRNAs targeting HER2 . MKN45 cells were transduced with control (sh_ Luc ) or with HER2 shRNAs (sh_ HER2 #1 and #2) and cultured in the presence of 3 μM doxycycline. Twenty-four hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of control vector-transduced cells (n = 3). ( e ) HER2 -depletion-mediated dephosphorylation of HER2. Non-depleted and HER2 -depleted MKN45 cells were treated as in ( d ). Forty-eight hours after treatment, cell lysates were processed for immunoblotting. ( f ) Growth curves of MKN45 cells transduced with control (sh_ Luc ) or with HER2 shRNAs (sh_ HER2 #1 and #2) in the presence of 3 μM doxycycline (n = 5). ( g , h ) Immunoprecipitation assay showed SOS1-HER2 complex in MKN45 cells. Data are mean ± SEM values. *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed Student’s t-test.

Article Snippet: Anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology).

Techniques: Transduction, Control, Cell Culture, Quantitative RT-PCR, Plasmid Preparation, De-Phosphorylation Assay, Western Blot, Immunoprecipitation, Two Tailed Test

Journal: Scientific Reports

Article Title: RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

doi: 10.1038/s41598-018-24969-w

Figure Lengend Snippet: RUNX1 directly transactivates SOS1 expression. ( a ) Proximal regulatory region (−1000 bp to +200 bp relative to TSS) of SOS1 . ( b ) Result of the ChIP analysis in MKN45 cells using anti-RUNX1 antibody, an isotype-matched control IgG and anti-Histone H3 antibody. ChIP products were amplified by PCR to determine the abundance of the indicated amplicons. ( c ) Luciferase reporter assay with SOS1 promoter. HEK293T cells were stably-transduced with the lentivirus expressing RUNX1 ( RUNX1 O/E) or control, together with the reporter vector expressing luciferase gene under SOS1 promoter. Cells were incubated with 3 μM doxycycline for 48 hours, then the luciferase activity was monitored by a luminometer. Result was normalized to that of the control sample (n = 3). Data are mean ± SEM values. ***P < 0.001, by two-tailed Student’s t-test.

Article Snippet: Anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology).

Techniques: Expressing, Control, Amplification, Luciferase, Reporter Assay, Stable Transfection, Transduction, Plasmid Preparation, Incubation, Activity Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

doi: 10.1038/s41598-018-24969-w

Figure Lengend Snippet: Anti-tumor effect of RUNX1 inhibition is SOS1-dependent. ( a ) Immunoblotting of RUNX1, SOS1 and GAPDH in non-depleted and RUNX1 -depleted ( RUNX1 K/D) NUGC4 and MKN45 cells transduced with or without lentivirus expressing SOS1 ( SOS1 O/E). Cells were treated with 3 μM doxycycline for 48 hours, then lysed for immunoblotting. ( b ) Restoring SOS1 expression in RUNX1 -depleted NUGC4 and MKN45 cells reverts RUNX1 -depletion-mediated growth inhibition. The indicated cells were cultured in the presence of 3 μM doxycycline (n = 5). Data are mean ± SEM values. **P < 0.01, by two-tailed Student’s t-test.

Article Snippet: Anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology).

Techniques: Inhibition, Western Blot, Transduction, Expressing, Cell Culture, Two Tailed Test

Journal: Scientific Reports

Article Title: RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

doi: 10.1038/s41598-018-24969-w

Figure Lengend Snippet: RUNX inhibitor Chb-M’ down-regulates ErbB2/HER2 pathway through attenuating SOS1 expression in gastric cancer cells. ( a ) Relative densitometric quantification of phospho-RTK array spots in Chb-M’-treated MKN45 cells compared to the control. Cells were treated with DMSO or 1 μM Chb-M’ for 48 hours, then the cells were lysed for the phospho-RTK array. Each receptor was spotted in duplicates (see Supplementary Fig. for the immunoblot image). ( b ) Down-regulation of SOS1 expression in Chb-M’-treated NUGC4 and MKN45 cells. Cells were treated with the indicated concentrations of Chb-M’. Six hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of DMSO-treated control cells (n = 3). ( c ) Down-regulation of SOS1 and dephosphorylation of HER2 in Chb-M’-treated NUGC4 and MKN45 cells. Cells were treated as in ( b ). Forty-eight hours after treatment, cell lysates were prepared and subjected to immunoblotting. Data are mean ± SEM values. *P < 0.05, **P < 0.01, n.s.; not significant, by two-tailed Student’s t-test.

Article Snippet: Anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology).

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, De-Phosphorylation Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

doi: 10.1038/s41598-018-24969-w

Figure Lengend Snippet: Chb-M’-mediated anti-tumor effect in gastric cancer cells. ( a , b ) Immunohistochemistry images of SOS1 expression in the MKN45-derived tumor treated with Chb-M’ or control DMSO. NOG mice were subcutaneously-transplanted with MKN45 cell at Day 1. Treatments either by Chb-M’ (320 μg/kg body weight, twice a week, i.v.) or equivalent amount of DMSO was continued from Day 8 until Day 21, then the mice were properly-sacrificed for tumor resection. Resected tumors were fixed with 10% formaldehyde for immunohistochemistry assay. Scale bars 200 μm for low magnification and 25 μm for high magnification (a). SOS1 staining in the MKN45-derived tumor treated with DMSO or Chb-M’ was quantified by Image J (b). ( c ) Dose-response curves of Chb, Chb-S and Chb-M’ in NUGC4 and MKN45 cells. Cells were treated with the indicated concentrations of PI polyamides or Chb. Forty-eight hours after treatment, cell viability was examined by WST assay (n = 5). ( d ) Dose-response curves of lapatinib and Chb-M’ in NUGC4 and MKN45 cells. Cells were treated as in (c). Forty-eight hours after treatment, cell viability was examined by WST assay (n = 5). Data are mean ± SEM values. *P < 0.05, **P < 0.01, ***P < 0.001, n.s.; not significant, by two-tailed Student’s t-test.

Article Snippet: Anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology).

Techniques: Immunohistochemistry, Expressing, Derivative Assay, Control, Staining, WST Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

doi: 10.1038/s41598-018-24969-w

Figure Lengend Snippet: SOS1 -overexpression confers resistance to the Chb-M’ treatment in gastric cancer cells. ( a ) Immunoblotting of SOS1 and GAPDH in NUGC4 and MKN45 cells transduced with control or SOS1 -expressing lentivirus. Cells were treated with 3 μM doxycycline for 48 hours to induce SOS1 expression, then lysed for immunoblotting. ( b ) Dose-response curves of Chb-M’ in NUGC4 and MKN45 cells transduced either with the lentivirus expressing SOS1 ( SOS1 O/E) or control. Cells were simultaneously treated with 3 μM doxycycline and various concentrations of Chb-M’. Forty-eight hours after treatment, cell viability was examined by WST assay (n = 5). ( c ) IC50 values of Chb-M’ in NUGC4 and MKN45 cells transduced either with the lentivirus expressing SOS1 ( SOS1 O/E) or control. Cells were treated as in (b) (n = 5). Data are mean ± SEM values. *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed Student’s t-test.

Article Snippet: Anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology).

Techniques: Over Expression, Western Blot, Transduction, Control, Expressing, WST Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: MicroRNA-448 suppresses osteosarcoma cell proliferation and invasion through targeting EPHA7

doi: 10.1371/journal.pone.0175553

Figure Lengend Snippet: (A) The expression level of miR-448 in the osteosarcoma tissues and their related normal tissues was determined by qRT-PCR. (B) The expression level of miR-448 was lower in osteosarcoma tissues compared to that in the related normal tissues.

Article Snippet: Human osteosarcoma cell lines (U2OS, MG-63, SAOS-2 and SOSP-9607) and an osteoblast cell line (hFOB) were obtained from the American Type Culture Collection and cultured in the DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum). miR-448 mimic and scramble mimic, EPHA7 vector and control vector were purchased from Dharmacon.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: MicroRNA-448 suppresses osteosarcoma cell proliferation and invasion through targeting EPHA7

doi: 10.1371/journal.pone.0175553

Figure Lengend Snippet: (A) The expression level of EPHA7 in osteosarcoma tissues and their related normal tissues was measured by qRT-PCR. (B) The expression level of EPHA7 was higher in the osteosarcoma tissues compared to that in the related normal tissues. (C) The expression level of EPHA7 was inversely correlated with that of miR-448 in osteosarcoma tissues.

Article Snippet: Human osteosarcoma cell lines (U2OS, MG-63, SAOS-2 and SOSP-9607) and an osteoblast cell line (hFOB) were obtained from the American Type Culture Collection and cultured in the DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum). miR-448 mimic and scramble mimic, EPHA7 vector and control vector were purchased from Dharmacon.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: MicroRNA-448 suppresses osteosarcoma cell proliferation and invasion through targeting EPHA7

doi: 10.1371/journal.pone.0175553

Figure Lengend Snippet: (A) The expression level of miR-448 in osteosarcoma cell lines (U2OS, MG-63, SAOS-2 and SOSP-9607) and the osteoblast cell line (hFOB) was determined by qRT-PCR. (B) miR-448 expression was significantly upregulated in the MG-63 cells after treatment with miR-448 mimic. (C) Elevated expression of miR-448 suppressed MG-63 cell proliferation. (D) Overexpression of miR-448 also decreased cyclin D1 expression in the MG-63 cells.(E) miR-448 expression was significantly upregulated in the U2OS cells after treatment with miR-448 mimic.(F) Elevated expression of miR-448 suppressed U2OS cell proliferation.(G) Overexpression of miR-448 inhibited MG-63 cell colony formation. The relative cell colony formation is shown. (H) Overexpression of miR-448 inhibited U2OS cell colony formation. The relative cell colony formation is shown. (I) Ectopic expression of miR-448 suppressed MG-63 cell migration. The relative open wound is shown. (J)Ectopic expression of miR-448 suppressed U2OS cell migration. The relative open wound is shown.*p<0.05, **p<0.01 and ***p<0.001.

Article Snippet: Human osteosarcoma cell lines (U2OS, MG-63, SAOS-2 and SOSP-9607) and an osteoblast cell line (hFOB) were obtained from the American Type Culture Collection and cultured in the DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum). miR-448 mimic and scramble mimic, EPHA7 vector and control vector were purchased from Dharmacon.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Migration

Journal: PLoS ONE

Article Title: MicroRNA-448 suppresses osteosarcoma cell proliferation and invasion through targeting EPHA7

doi: 10.1371/journal.pone.0175553

Figure Lengend Snippet: (A) The expression level of EPHA7 in the osteosarcoma cell lines (U2OS, MG-63, SAOS-2 and SOSP-9607)and osteoblast cell line (hFOB) was measured by qRT-PCR. (B) The EPHA7 mRNA expression was significantly upregulated in the MG-63 cells after treatment with EPHA7 vector. (C) The protein expression of EPHA7 was determined by Western blot. (D) CCK8 assay results demonstrated that EPHA7 overexpression restored miR-448 overexpression MG-63 cell proliferation. (E) Overexpression of EPHA7 promoted cyclin D1 expression in the miR-448 overexpressing MG-63 cells. (F) Overexpression of EPHA7 promoted miR-448 overexpressing MG-63 cell migration. (G) The relative migrative wound was shown. *p<0.05.

Article Snippet: Human osteosarcoma cell lines (U2OS, MG-63, SAOS-2 and SOSP-9607) and an osteoblast cell line (hFOB) were obtained from the American Type Culture Collection and cultured in the DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum). miR-448 mimic and scramble mimic, EPHA7 vector and control vector were purchased from Dharmacon.

Techniques: Expressing, Quantitative RT-PCR, Plasmid Preparation, Western Blot, CCK-8 Assay, Over Expression, Migration

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) FSS causes the rapid loss of sclerostin protein through a number of molecular mediators. ( B ) Ocy454 cells (n = 3–4) or ( C ) UMR106 cells (n = 3) were exposed to 1 min of FSS at 4 dynes/cm 2 and lysed 5 min post-flow. Western blots were probed for sclerostin, GAPDH, pCaMKII, and total CaMKII. ( D ) Sixteen week old female C57Bl/6 mice were ulnar loaded (1800 με, 90 s, 2 Hz), cortical osteocyte-enriched lysates isolated 5 min post-load, and western blots probed for sclerostin (n = 10 mice), pCaMKII, and total CaMKII (n = 5 mice). Sclerostin abundance relative to the loading control or pCaMKII relative to total CaMKII was quantified. ( E ) Ocy454 cells with endogenous sclerostin (n = 2), ( F ) UMR106 cells with endogenous sclerostin (n = 4), or ( G ) Ocy454 cells transfected with Myc-tagged sclerostin (n = 1) were subjected to 5 min of FSS at 4 dynes/cm 2 and lysed at the indicated times post-flow. Western blots were probed for sclerostin and β-actin. A representative time course is shown for each. Sclerostin abundance relative to the loading control was quantified. For each antibody, western blots are from a single gel and exposure; a vertical black line indicates removal of irrelevant lanes. Graphs depict mean ± SD. *p<0.05, **p<0.01 by unpaired two-tailed t-tests ( B–D ).

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Western Blot, Isolation, Transfection, Two Tailed Test

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Seventeen week old male mice were subjected to a single bout of ulnar loading (1800 με), and ulnae were fixed in warm formalin 5 min after loading. Cryosections were stained for sclerostin to identify sclerostin-positive osteocytes, which were counted and presented as a proportion of total osteocytes in a selected ROI (n = 6). Yellow arrows indicate sclerostin-negative osteocytes, defined by the presence of DAPI staining but without detectable sclerostin. Yellow asterisks indicate non-specific staining. ( B ) Ocy454 cells transfected with GFP-sclerostin were subjected to 5 min of FSS at 4 dynes/cm 2 and lysed immediately post-flow. Western blots were probed for sclerostin and β-actin. ( C ) Ocy454 cells were subjected to 5 min of FSS at 4 dynes/cm 2 and lysed immediately post-flow. Western blots were probed for pro-collagen type 1α1 and β-actin. ( D, E ) Ocy454 cells were treated with PTH (10 nM) for 30 min and lysed. Western blots were probed for sclerostin, β-actin, pro-collagen type 1α1, and α-tubulin.

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Staining, Transfection, Western Blot

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Ocy454 cells transfected with GFP-sclerostin were treated with vehicle or PTH (1–34) (10 nM) for the indicated time and were lysed. Western blots were probed for sclerostin and β-actin (n = 2–3). ( B ) Dissected tibiae flushed of marrow were treated with vehicle or PTH (1–34) (10 nM) for 30 min ex vivo and homogenized. Western blots were probed for sclerostin and β-actin (n = 6 mice). ( C ) Ocy454 cells were treated with vehicle or PTH (1–34) (10 nM) for the indicated time and were lysed. Western blots were probed for pCaMKII and total CaMKII (n = 6–8). Graphs depict mean ± SD. *p<0.05, **p<0.01 by two-way ANOVA with Holm–Sidak post hoc correction ( A, C ) or unpaired two-tailed t-test ( B ).

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Transfection, Western Blot, Ex Vivo, Two Tailed Test

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: Ocy454 cells were treated with PTH (10 nM) for 30 min and then collected 5.5 hr after treatment to examine Sost mRNA levels as normalized to Gapdh , Rpl13 , and Hprt by RT-qPCR (n=6).

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Quantitative RT-PCR

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Ocy454 cells transfected with GFP-sclerostin were treated with cycloheximide (150 µg/mL) to prevent new protein synthesis and either DMSO (0.1%), bafilomycin A1 (100 nM) to inhibit lysosomal degradation, brefeldin A (2 μm) to inhibit secretion, or MG-132 (10 μm) to inhibit the proteasome 4 hr prior to FSS. Cells were subjected to 5 min of FSS at 4 dynes/cm 2 and lysed immediately after the end of FSS or 30 min after the conclusion of FSS. Western blots were probed for sclerostin and β-actin. Time courses show mean ± SEM (n = 3–6 independent experiments/group). ( B ) Ocy454 cells transfected with GFP-sclerostin were pre-treated with DMSO (0.1%) or bafilomycin A1 (100 nM) to inhibit lysosomal degradation for 30 min prior to the addition of vehicle or PTH (1–34) (10 nM) for an additional 30 min (n = 3). Sclerostin abundance relative to the loading control was quantified. Graph depicts mean ± SD. *p<0.05, **p<0.01, ****p<0.0001 by two-way ANOVA with Holm–Sidak post hoc correction. ( C ) Amino acid sequences for sclerostin from mouse, human, rat, cow, and chicken were aligned using NCBI COBALT. Abbreviated sequences are shown and are annotated for putative lysosomal signal sequences. Full sequences are presented in .

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Transfection, Western Blot

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) UMR106 cells were treated with cycloheximide (150 µg/mL) and either DMSO (0.1%), bafilomycin A1 (100 nM) for 6 hr, or leupeptin (200 μM) for 4 hr prior to FSS. Cells were subjected to 1 min of FSS at 4 dynes/cm 2 and were lysed 5 or 10 min after the conclusion of FSS. Western blots were probed for sclerostin. Sclerostin abundance relative to total protein was quantified. For each antibody, blots are from a single gel and exposure; a vertical black line indicates removal of irrelevant lanes. ( B ) UMR106 cells were treated with cycloheximide (150 µg/mL), and lysates were collected at 0, 1, 2, and 4 hr after treatment in the absence of stimuli. Whole-cell lysates were western blotted for sclerostin abundance (n = 2). Sclerostin abundance relative to the total protein was quantified. Graph shows mean ± SD and best-fit linear regression constrained through y = 1. ( C ) Full amino acid sequences for sclerostin from mouse, human, rat, cow, and chicken were aligned using NCBI COBALT. Putative lysosomal signal sequences are annotated. The secretory signal peptide is annotated for amino acids 1–23.

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Western Blot

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Endogenous sclerostin (top) and GFP-tagged sclerostin (bottom) form discrete puncta in Ocy454 cells. ( B ) Ocy454 cells were transfected with GFP-sclerostin, and lysosomes were visualized with Lysotracker (1 mM, 1 hr) or siR-Lysosome (1 μM, 4 hr). Scale bar represents 10 μm. ( C ) Ocy454 cells were stained for endogenous sclerostin and either p62/sequestosome-1 or Rab27a to evaluate co-localization with these lysosome-associated proteins. ( D ) Ocy454 cells were exposed to 1 min of FSS at 4 dynes/cm 2 , lysed immediately post-flow, and western blotted for p62/sequestosome-1, β-actin, and LC3 (n = 4). ( E ) Ocy454 cells were treated with PTH (1–34) (10 nM) for 5 min, lysed, and western blotted for p62/sequestosome-1 and β-actin (n = 4) and LC3 (n = 8). ( F ) UMR106 cells were subjected to FSS for 5 min, then Magic Red Cathepsin B was applied for 10 min, fixed, and imaged to assess lysosome activity (n = 9). ( G ) Ocy454 cells were treated with DMSO or KN-93 (10 μM) to inhibit CaMKII for 1 hr prior to FSS at 4 dynes/cm 2 for 5 min before lysing immediately after FSS. Western blots were probed for p62/sequestosome-1 and β-actin (n = 3). ( H ) Ocy454 cells were transfected with a plasmid expressing either GFP or dominant negative CaMKII T286A prior to treatment with PTH (1–34) (10 nM) for 30 min. Western blots were probed for p62/sequestosome-1 and β-actin (n = 3). Graphs depict mean ± SD. *p<0.05, **p<0.01 by unpaired two-tailed t-test ( D–F ) or by two-way ANOVA with Holm–Sidak post hoc correction ( G, H ).

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Transfection, Staining, Western Blot, Activity Assay, Plasmid Preparation, Expressing, Dominant Negative Mutation, Two Tailed Test

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: GFP-sclerostin-transfected and siR-lysosome-stained Ocy454 cells were imaged in Z-stacks with 0.5 μm steps and reconstructed into 3D images. Nikon general analysis was programmed to identify lysosomes containing sclerostin. GFP-sclerostin is pseudocolored in pink, siR-lysosome in yellow, and lysosomes containing sclerostin in cyan. Z-stack images from GFP-sclerostin- and siR-lysosome-labeled cells were analyzed for co-localization coefficients. M1 represents the overlap of sclerostin with siR-lysosome; M2 represents the overlap of siR-lysosome with sclerostin (n = 3).

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Transfection, Staining, Labeling

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Ocy454 cells transfected with GFP-sclerostin were treated with vehicle (water) or 10 μM SNAP, a nitric oxide donor, and lysed after 5 min. Western blots were probed for sclerostin, α-tubulin, pCaMKII, and total CaMKII (n = 3). For each antibody, blots are from a single gel and exposure; a vertical black line indicates removal of irrelevant lanes. ( B ) Ocy454 cells transfected with myc-tagged sclerostin were treated with DMSO or bafilomycin A1 (100 nM) to inhibit lysosomal degradation, for 30 min, then treated with SNAP, a nitric oxide donor, for 5 min and lysed. Western blots were probed for sclerostin and α-tubulin (n = 2–3). ( C ) UMR106 cells were treated with vehicle or L-NAME (1 mM) to inhibit nitric oxide synthases (NOSs) for 1 hr and then exposed to 1 or 5 min of FSS. Lysates from cells exposed to 1 min of FSS were probed for p62/sequestosome-1 and α-tubulin abundance and lysates from cells exposed to 5 min of sclerostin were probed for sclerostin and α-tubulin abundance (n = 3). Graphs depict mean ± SD. *p<0.05, **p<0.01, ***p<0.001 by unpaired two-tailed t-test ( A ) or two-way ANOVA with Holm–Sidak post hoc test ( B , C ).

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Transfection, Western Blot, Two Tailed Test

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) UMR106 cells transfected with KillerRed imaged before and after stimulation with LED light. DCF was used to track ROS production. ( B ) UMR106 cells transfected with KillerRed were stimulated with LED light for 5 min and lysed 5 min after. Westerns were probed for sclerostin, GAPDH, pCaMKII, and total CaMKII.

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Transfection

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Dissected ulnae and radii flushed of marrow were treated with hydrogen peroxide (100 μM) as a source of ROS for 5 min before homogenization. Western blots were probed with sclerostin and β-actin (n = 6 mice). ( B ) Thirteen week old male C57Bl/6 mice treated with vehicle (saline, n = 10 mice) or apocynin (3 mg/kg, n = 8 mice) to inhibit NOX2 were forearm loaded (1800 με, 90 s, 2 Hz) and labeled with calcein and alizarin red at the indicated times for dynamic histomorphometry. Representative periosteal double labeling is shown. ( C ) Periosteal bone formation rate (Ps.BFR) and ( D ) periosteal mineral apposition rate (Ps.MAR) were calculated. ( E ) Fourteen to 17 week old male and female C57Bl/6 mice treated with vehicle (saline + 4% DMSO, i.p., n = 14) or apocynin (3 mg/kg in saline, i.p., n = 12 mice) to inhibit NOX2 were treated 2 hr prior to ulnar loading (2000 με, 90 s, 2 Hz). Non-loaded and loaded limbs were isolated 5 min post-load, and western blots were probed for sclerostin and β-actin. Vehicle data is duplicated in as all animals were run and processed together. Graphs depict mean ± SD. *p<0.05, **p<0.01 by unpaired two-tailed t-test ( A ), two-way ANOVA with Holm–Sidak post hoc correction ( C, D ), or Kruskal–Wallis with Dunn’s post hoc correction ( E ).

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Homogenization, Western Blot, Labeling, Isolation, Two Tailed Test

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet: ( A ) Fifteen week old male C57Bl/6 mice treated with vehicle (saline + 4% DMSO, n = 7 mice) or bafilomycin A1 (1 mg/kg, n = 7 mice) to inhibit lysosomal degradation were forearm loaded (2000 με, 90 s, 2 Hz) and labeled with calcein and alizarin red at the indicated times for dynamic histomorphometry. Representative periosteal double labeling are shown. ( B ) Periosteal bone formation rate (Ps.BFR) and ( C ) periosteal mineral apposition rate (Ps.MAR) were calculated. ( D ) Fourteen to 17 week old male and female C57Bl/6 mice treated with vehicle (saline + 4% DMSO, i.p., n = 14) or bafilomycin A1 (1 mg/kg in saline + 4% DMSO, i.p., n = 12 mice) to inhibit lysosomal degradation were treated 2 hr prior to ulnar loading (2000 με, 90 s, 2 Hz). Non-loaded and loaded limbs were isolated 5 min post-load, and western blots were probed for sclerostin and β-actin. Vehicle data is duplicated in as all animals were run and processed together. ( E ) Human iPSC-derived osteoblasts from either control (non-diseased) or Gaucher disease patients were treated with vehicle or recombinant glucocerebrosidase (rGCase, 0.24 U/mL) for 5 days, then lysed for western blotting. Western blots were probed for sclerostin and GAPDH (n = 3 independent patient-derived iPSC lines/group). Graphs depict mean ± SD. *p<0.05, **p<0.01 by two-way ANOVA with Holm–Sidak post hoc correction ( B , C , and E ) or Kruskal–Wallis with Dunn’s post hoc correction ( D ). ( F ) FSS causes the rapid degradation of sclerostin protein by the lysosome through a number of molecular mediators. PTH, converging with this FSS mechano-transduction pathway at CaMKII, also mediates the lysosomal degradation of sclerostin protein. Icons outlined red are molecular mechanisms controlling sclerostin abundance that have been described within this manuscript that integrate into our previously described mechano-transduction cascade. Osteoanabolic stimuli, working through reactive oxygen (ROS) and reactive nitrogen species (RNS), direct sclerostin to the lysosome for degradation. This results in reduced sclerostin to allow for bone formation. PM: plasma membrane; ROS: reactive oxygen species; NO: nitric oxide.

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Labeling, Isolation, Western Blot, Derivative Assay, Recombinant, Transduction

Journal: eLife

Article Title: Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein

doi: 10.7554/eLife.64393

Figure Lengend Snippet:

Article Snippet: Transfected construct ( Mus musculus ) , myc-tagged sclerostin , Origene , #MR222588 , .

Techniques: Transfection, Construct, Sequencing, In Vitro, In Vivo, Software, Staining

Journal: bioRxiv

Article Title: Activity of Estrogen Receptor β Agonists in Therapy-Resistant Estrogen Receptor-Positive Breast Cancer

doi: 10.1101/2022.01.14.476328

Figure Lengend Snippet: A and B , Expression of ESR1 and ESR2 in immortalized mammary MCF10A, transformed ERα+ MCF7 and T47D, endocrine resistant MCF7-TamR, MCF7-FasR, T47D-TamR, and T47D-FasR, CDK6 over-expressing MCF7 (MCF7-CDK6 O/E), CDK4/6 inhibitor resistant MCF7 (MCF7-CDK4/6iR) and T47D (T47D-CDK4/6iR), ZR-75-1, and triple negative breast cancer (TNBC; MDA-MB231, MDA-MB-468, Hs578t) cell lines. Total RNA was isolated from the established cell lines using TRIzol. The expression of each gene was assessed by quantitative RT-PCR (qRT-PCR) performed with the DNase-treated RNA samples using gene-specific primers spanning exon-exon junctions that include large introns in the corresponding genomic sequence to avoid genomic DNA amplification. Gene expression was calculated by ΔΔCt method using GAPDH as an internal control. The expression of each gene is shown as the fold change relative to MCF10A. All reactions were done in triplicate and the experiment was repeated twice. Data were plotted as mean ± SD. A , ESR2 genes; full length (left) and all isoforms (right). B , ESR1 . C , whole-cell lysates were extracted and immunoblot analyses were performed for ERβ and GAPDH (loading control) (upper panel), and ERα and GAPDH (lower panel). Intensity of the protein bands was quantified using Image Studio (LiCor) software. Numbers under the lanes of each cell line represent normalized values of the corresponding protein band (ERβ or ERα). Normalized band intensity of MCF10A was considered as 1. Immunoblot analyses were repeated twice with corresponding biological replicates. Reproducible results were obtained in each independent experiment. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. For ERβ (upper panel) two different exposures were provided; low exp.= low exposure; high exp.= higher exposure of the blot D, ERE-Luciferase driven promoter activity upon treatment with selective ERβ agonists is significantly higher in ectopically expressing cells with ERβ compared to that of ERα . HEK293T cells were transfected with c-Flag pcDNA3 (vector control), c-Flag ERα or c-Flag ERβ in combination with ERE-Luciferase (reporter) and TK-renilla (pRLTK; internal control) plasmids (as described in Materials and Methods section). Forty eight hours after treatment of the cells with ERβ specific agonists Renilla and Firefly luciferase activities were measured using the dual-luciferase reporter assay system. Renilla luciferase was normalized to Firefly luciferase. Treatment with: OSU-ERb-12 (0-10 μmol/L) (left) and LY500307 (0-10 μmol/L) (middle). Each assay was performed in triplicate with three experimental replicates. (mean + SD, *: p<0.05, **: p<0.01). Right panel shows equal expression of ERα and ERβ as determined by western blot analysis using anti-flag antibody. Intensity of Flag-ERα/ERβ was normalized to GAPDH. The numbers under the corresponding protein band represent normalized values of the corresponding protein band intensity.

Article Snippet: Plasmids expressing pcDNA3 (OHu23619C; pcDNA3.1+: RRID: Addgene_10842), ERβ (OHu25562C; pcDNA3.1+), c-Flag pcDNA3 (OHu23619D; pcDNA3.1+/C-(K) DYK), c-Flag ERα (OHu26586D; pcDNA3.1+/C-(K) DYK), and c-Flag ERβ (OHu25562D; pcDNA3.1+/C-(K) DYK were obtained from GenScript.

Techniques: Expressing, Transformation Assay, Isolation, Quantitative RT-PCR, Sequencing, DNA Amplification, Gene Expression, Control, Western Blot, Software, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Reporter Assay

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Fig. 1. Targeted disruption of the murine mSos1 gene in ES cells and mice. (A) Schematic representation of the mSos1 locus and targeting vector. Boxes in the wild-type allele schematics represent the exons of the mSos1 CDC25–H domain. The open boxes in the targeting vector schematics represent the pgk-neo and pgk-tk selectable markers. The position of the 3′–flanking probe used in Southern blotting is indicated. (B) Homologous recombination of the targeting vector in ES cells was verified by Southern blotting, digesting genomic DNA with EcoRV and hybridizing with the 3′–flanking probe. The wild-type allele produced a 14 kb band, whereas the mutant allele yielded a 6.9 kb band due to the introduction of a new EcoRV site in the targeting vector. (C) Genotyping of embryos arising from heterozygous crosses was performed by RT–PCR using the oligonucleotides indicated, whose sequences are given in Materials and methods. The LM87 and LM107 primers are specific for the sos1 gene and amplify a fragment of 612 bp. The LM82 primer is specific for the Neo-PGK promoter and amplifies a fragment of 410 bp with LM87.

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques: Plasmid Preparation, Southern Blot, Homologous Recombination, Produced, Mutagenesis, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Fig. 2. Placental labyrinth defects in sos1–/– animals. The left column (A–C) displays control preparations of mouse wild-type sos1 (+/+) placental/embryo structures at DPC12 (H&E-stained) at low, medium and high magnification, respectively. The right column (D–F) shows the corresponding sos1–/– preparations at DPC12. The wild type (A) contains embryo and developed placenta, while –/– (D) shows hemorrhage (dark red areas) into the labyrinth caused by the collapse of the labyrinth structure, and a dead embryo (not visible). The wild type (B) shows the chorionic plate (pale zone at top of figure), labyrinth, prominent spongiotrophoblast layer, giant cells and decidual layers, while –/– (E) has a thinner labyrinth, and it is difficult to discern the spongiotrophoblast layer. The wild type (C) shows a normal labyrinth, with maternal erythrocytes (M), labyrinth trophoblasts (LTB) and strings of embryonic nucleated erythrocytes within embryonic blood vessels. The –/– (F) has fewer nucleated embryonic erythrocytes (arrows) within fewer embryonic blood vessels, dysplastic labyrinth trophoblasts and a multinucleated giant cell (indicated by the letter G).

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques: Staining

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Fig. 3. Sos1 and Sos2 expression in normal murine placental tissues. In situ hybridization (A) and histochemistry (B) of wild-type DPC12 placental tissues. (A) Left column: in situ hybridization with sos1 antisense (upper panel) and sense (negative control, lower panel) probes, ×50. Right column: in situ hybridization with sos2 antisense (upper panel) and sense (negative control, lower panel) probes. The results show a high sos1 mRNA signal in spongiotrophoblasts, less in labyrinth trophoblasts and none in the decidua. There was less sos2 expression. (B) Immunohistochemistry of normal placenta with Sos1- and Sos2-specific antibodies at low (a and c) and high (b and d) magnification, hematoxylin-stained. (a and b) The results show high expression of Sos1 protein within spongiotrophoblasts and less in the labyrinth. (c and d) Sos2 signal is seen in giant cell trophoblasts but not in spongiotrophoblasts and labyrinth trophoblasts. (e) Negative control of immunohistochemical staining using the same reagents except for the primary Sos1 and Sos2 antibodies (rabbit IgG).

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques: Expressing, In Situ Hybridization, Negative Control, Immunohistochemistry, Staining, Immunohistochemical staining

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Fig. 4. Analysis of signaling events in placenta. For (A) and (B), individual E10 placentas from Sos1+/– matings were dissected and separated into fetal portion (PF) and maternal portion (PM). (A) Placental ERK activity. Anti-ERK2 immunocomplexes were assayed for kinase activity using MBP as substrate. The middle and lower panels, with the control immunoblots, indicate that the extracts contained comparable amounts of ERK2 and EGFR protein, respectively. (B) Complexes between Sos1 and phosphorylated RTKs and other tyrosine-phosphorylated proteins. Placental extracts were immunoprecipitated with Sos1-specific antibody followed by immunoblotting with anti-phosphotyrosine antibody (PY). (C) Sos1 versus Sos2 complexes with phosphorylated proteins. Extracts from the pooled fetal portions of several wild-type (+/+) E10 placentas were immunoprecipitated with anti-Sos-1 or anti-Sos2 antibodies followed by anti-PY blot. The lower part, with the reblots, shows strong Sos1 and Sos2 signals precipitated by the homologous antibody.

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques: Activity Assay, Western Blot, Immunoprecipitation

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Transforming activities induced by oncogenes

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques:

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Fig. 5. Reconstitution of Sos1 in –/– cells restores the transforming activity of v–Src. –/– cells were transiently transfected with empty vector or a plasmid expressing mouse Sos1 followed by infection with the v–Src or v–Ras virus. The photomicrographs are from the resulting lines. (A) –/– cells transfected with Sos1, not infected with virus (negative control); (B) –/– cells transfected with empty vector and infected with v–Src virus; (C) –/– cells transfected with empty vector and infected with v–Ras virus; and (D) –/– cells transfected with Sos1 and infected with v–Src virus.

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Infection, Negative Control

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Fig. 6. Ligand-induced acute signaling in wild-type and Sos1 –/– cells. (A) EGF-dependent changes in Ras⋅GTP. Subconfluent Sos1 +/+, +/– and –/– cells were deprived of serum for 16 h, then metabolically labeled with [32P]orthophosphate for 10 h and stimulated with 10 ng/ml EGF for 5 min. Cells were then lysed and analyzed for Ras⋅GTP and Ras⋅GDP (Zhang et al., 1992). The results represent the average of three experiments. (B) Ligand-induced ERK activity. Serum-deprived cells were treated with or without ligand for 5 min as indicated. Anti-ERK2 immunocomplexes were assayed for kinase activity using MBP as substrate. Data shown are representative of two experiments.

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques: Metabolic Labelling, Labeling, Activity Assay

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Fig. 7. Comparison of EGF-induced long-term ERK activity in Sos1 +/+ or +/– cells with Sos1-knockout cells. Sos1 +/+, +/– or –/– embryo cells were infected with virus encoding human EGFR. Cells were treated with EGF (10 ng/ml) for the time periods indicated. (A) A 100 μg aliquot of protein from Sos1 +/+ and –/– cell lysates was immunoprecipitated with anti-ERK2 antibody followed by immune complex kinase assay using MBP as a substrate. (B) Equal amounts of protein from EGF-treated Sos1 (–/–) or (+/+) embryo cells were analyzed by immunoblotting with the indicated antibody. (C) A 100 μg aliquot of protein from EGF-treated Sos1 +/– and –/– cell lysates was immunoprecipitated with anti-ERK2 antibody followed by immune complex kinase assay as in (A). The amount of radioactivity present in the phosphorylated MBP was quantitated by a phosphoimager, and the fold increase is shown underneath the figure. (D) The upper part of the gel in (C) was immunoblotted with anti-ERK2 antibody, as a loading control.

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques: Activity Assay, Knock-Out, Infection, Immunoprecipitation, Immune Complex Kinase Assay, Western Blot, Radioactivity

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Fig. 8. EGF-induced formation of signaling complexes in Sos1 +/+, +/– and –/– cells. EGFR virus-infected cells were serum starved and treated with EGF (10 ng/ml) for the periods indicated. Equal amounts of cell lysates were immunoprecipitated with antibodies against Sos–1 or Sos–2, followed by blotting with anti-phosphotyrosine antibody (PY) or GRB2. The anti-PY blots were then reprobed with anti-Sos antibodies after stripping to serve as loading controls. Comparison of Sos–1 or Sos–2 forming a complex with activated EGFR or GRB2 in (A) Sos1 +/+ versus –/– cells, and in (B) Sos1 +/– versus –/– cells.

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques: Infection, Immunoprecipitation, Stripping Membranes

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Fig. 9. Analysis of complex formation between Shc and Sos in Sos1 +/+, +/– and –/– cells. EGFR virus-infected cells were serum starved and treated with EGF (10 ng/ml) for the times indicated. Equal amounts of cell lysates were immunoprecipitated with polyclonal antibody against Shc followed by blotting with anti-Sos–1 or anti-Sos–2 antibodies. The lower panels used anti-Shc monoclonal antibody as loading controls. Comparison of Sos1 or Sos2 co-immunoprecipitated with Shc in (A) Sos1 +/+ versus –/– cells, and in (B) Sos1 +/– versus –/– cells.

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques: Infection, Immunoprecipitation

Journal:

Article Title: The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties

doi: 10.1093/emboj/19.4.642

Figure Lengend Snippet: Fig. 10. Analysis of complex formation in v–Src virus-infected Sos1 +/+ and –/–cells. Sos1 +/+ and –/– cells with or without v–Src virus infection were serum starved for 16–18 h. (A) Comparison of Sos1or Sos2 co-immunoprecipitated with Shc. Equal amounts of cell lysates were immunoprecipitated with anti-Shc antibody followed by blotting with anti-Sos1 or anti-Sos2 antibodies. The lower panels used anti-Shc monoclonal antibody as loading controls. Expression of Sos2 and Sos1 in v–Src virus-infected +/+ and –/– cells was examined by immunoblotting. (B) Comparison of Sos1 or Sos2 co-immunoprecipitated with v–Src. Equal amounts of the cell extracts were immunoprecipitated with anti-Src antibody followed by blotting with anti-Sos antibodies as indicated. Lysates were immunoprecipitated and blotted with anti-Src antibody to confirm v–Src expression in the infected cells.

Article Snippet: For reconstitution of Sos1 in –/– cells, pcDNA3 vector (Invitrogen) or pcDNA3-Sos1, which expressed wild-type Sos1, was transfected into –/– cells in 35 mm dishes, using calcium phosphate precipitation as described ( Willumsen et al., 1991 ).

Techniques: Infection, Immunoprecipitation, Expressing, Western Blot

Journal: Nature Communications

Article Title: Adipocytic sclerostin loop3-LRP4 interaction required by sclerostin to impair whole-body lipid and glucose metabolism

doi: 10.1038/s41467-026-68526-w

Figure Lengend Snippet: a The serum sclerostin levels in individuals before (Baseline) and after newly diagnosed T2DM (ND-T2DM). Statistical significance was calculated using paired t -test. b The serum sclerostin levels in postmenopausal osteoporosis (POP) patients with T2DM and POP patients without T2DM. Statistical significance was calculated using unpaired t -test. Note: Baseline: healthy individuals ( n = 119); ND-T2DM: newly diagnosed T2DM patients ( n = 119). Normal bone mass (NBM), normal glucose tolerance (NGT). Individuals without T2DM ( n = 9); individuals with T2DM ( n = 10); POP patients without T2DM ( n = 22); POP patients with T2DM ( n = 24). All data were expressed as mean ± SD. All tests were two-sided.

Article Snippet: As previously described by Kim et al., sclerostin overproduction was achieved by injecting adeno-associated viruses (AAV8-CMV) encoding mouse SOST (Vector Biolabs, AAV-272868) via the tail vein to 6-month-old male C57BL/6J mice for 6 weeks .

Techniques:

Journal: Nature Communications

Article Title: Adipocytic sclerostin loop3-LRP4 interaction required by sclerostin to impair whole-body lipid and glucose metabolism

doi: 10.1038/s41467-026-68526-w

Figure Lengend Snippet: a Body weights of wild-type (WT) mice, full-length sclerostin knock-in ( SOST ki ) mice, and loop3-deficient sclerostin knock-in ( Δloop3-SOST ki ) mice. b Fat pad weights in WT mice, SOST ki mice, and Δloop3-SOST ki mice. gWAT: gonadal white adipose tissue; iWAT: inguinal white adipose tissue; rpWAT: retroperitoneal white adipose tissue; iBAT: interscapular brown adipose tissue. c Serum free fatty acids in WT mice, SOST ki mice, and Δloop3-SOST ki mice. d Representative images of histological sections (left) and frequency distribution of adipocyte sizes (right) in gWAT and iWAT from WT mice, SOST ki mice, and Δloop3-SOST ki mice. Scale bars, 100 μm. ( e ) Expression levels of genes associated with lipid anabolism ( Acaca , Srebp2 , Lpl , and Fasn ) and catabolism ( Acads , Cpt1a and Acadvl ) in gWAT from WT mice, SOST ki mice and Δloop3-SOST ki mice detected by qPCR. f Fasting blood glucose in WT mice, SOST ki mice, and Δloop3-SOST ki mice. g Glucose tolerance test (GTT) (left), insulin tolerance test (ITT) (middle), and area under the curve (AUC) analysis for GTT and ITT (right) in WT mice, SOST ki mice, and Δloop3-SOST ki mice. h Expression levels of genes associated with glucose metabolism in gWAT from WT mice, SOST ki mice, and Δloop3-SOST ki mice detected by qPCR. Note: n = 7 biologically independent samples per group. All data were expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 for intergroup comparison (black * : WT vs. SOST ki ; orange * : SOST ki vs. Δloop3-SOST ki ). Statistical significance was calculated using unpaired t -test. ns: no significance. All tests were two-sided.

Article Snippet: As previously described by Kim et al., sclerostin overproduction was achieved by injecting adeno-associated viruses (AAV8-CMV) encoding mouse SOST (Vector Biolabs, AAV-272868) via the tail vein to 6-month-old male C57BL/6J mice for 6 weeks .

Techniques: Knock-In, Expressing, Comparison

Journal: Nature Communications

Article Title: Adipocytic sclerostin loop3-LRP4 interaction required by sclerostin to impair whole-body lipid and glucose metabolism

doi: 10.1038/s41467-026-68526-w

Figure Lengend Snippet: a Experimental design. b Body weights of SOST ki mice with or without Apc001OA treatment. c Fat pad weights in SOST ki mice with or without Apc001OA treatment. gWAT: gonadal white adipose tissue; iWAT: inguinal white adipose tissue; rpWAT: retroperitoneal white adipose tissue; iBAT: interscapular brown adipose tissue. d Serum free fatty acids in SOST ki mice with or without Apc001OA treatment. e Representative images of histological sections (left) and frequency distribution of adipocyte sizes (right) in gWAT and iWAT from SOST ki mice with or without Apc001OA treatment. Scale bars, 100 μm. f Expression levels of genes associated with lipid anabolism ( Acaca , Srebp2 , Lpl and Fasn ) and catabolism ( Acads , Cpt1a , and Acadvl ) in gWAT from SOST ki mice with or without Apc001OA treatment detected by qPCR. g Fasting blood glucose in SOST ki mice with or without Apc001OA treatment. h Glucose tolerance test (GTT) (left), insulin tolerance test (ITT) (middle), and area under the curve (AUC) analysis for GTT and ITT (right) in SOST ki mice with or without Apc001OA treatment. i Expression levels of genes associated with glucose metabolism in gWAT from SOST ki mice with or without Apc001OA treatment detected by qPCR. Note: n = 7 biologically independent samples per group. All data were expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 for intergroup comparison. Statistical significance was calculated using unpaired t -test. ns no significance. All tests were two-sided. Mouse image was created in BioRender. Zhang, G. (2025) https://BioRender.com/17da8dl .

Article Snippet: As previously described by Kim et al., sclerostin overproduction was achieved by injecting adeno-associated viruses (AAV8-CMV) encoding mouse SOST (Vector Biolabs, AAV-272868) via the tail vein to 6-month-old male C57BL/6J mice for 6 weeks .

Techniques: Expressing, Comparison

Journal: Nature Communications

Article Title: Adipocytic sclerostin loop3-LRP4 interaction required by sclerostin to impair whole-body lipid and glucose metabolism

doi: 10.1038/s41467-026-68526-w

Figure Lengend Snippet: a The schematic diagram of construction strategy and experimental design. b Body weights of SOST loop3m + HFD and SOST WT + HFD mice. c Food intake in SOST loop3m + HFD and SOST WT + HFD mice. Two-way ANOVA with Sidak’s multiple comparisons tests were conducted to evaluate intergroup variations. d Fat pad weights in SOST loop3m + HFD and SOST WT + HFD mice. gWAT: gonadal white adipose tissue; iWAT: inguinal white adipose tissue; rpWAT: retroperitoneal white adipose tissue; iBAT: interscapular brown adipose tissue. e Serum free fatty acids in SOST loop3m + HFD and SOST WT + HFD mice. f Representative images of histological sections (left) and frequency distribution of adipocyte sizes (right) in gWAT and iWAT from SOST loop3m + HFD and SOST WT + HFD mice. Scale bars, 200 μm. g Expression levels of genes associated with lipid anabolism ( Acaca , Srebp2 , Lpl and Fasn ) and catabolism ( Acads , Cpt1a ,and Acadvl ) in gWAT from SOST loop3m + HFD and SOST WT + HFD mice detected by qPCR. h Fasting blood glucose in SOST loop3m + HFD and SOST WT + HFD mice. i Glucose tolerance test (GTT) (left), insulin tolerance test (ITT) (middle), and area under the curve (AUC) analysis for GTT and ITT (right) in SOST loop3m + HFD and SOST WT + HFD mice. j Expression levels of genes associated with glucose metabolism in gWAT from SOST loop3m + HFD and SOST WT + HFD mice detected by qPCR. Note: n = 8 biologically independent samples per group. All data were expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 for intergroup comparison. Statistical significance was calculated using unpaired t -test. ns no significance. All tests were two-sided. Mouse image was created in BioRender. Zhang, G. (2025) https://BioRender.com/17da8dl .

Article Snippet: As previously described by Kim et al., sclerostin overproduction was achieved by injecting adeno-associated viruses (AAV8-CMV) encoding mouse SOST (Vector Biolabs, AAV-272868) via the tail vein to 6-month-old male C57BL/6J mice for 6 weeks .

Techniques: Expressing, Comparison

Journal: Nature Communications

Article Title: Adipocytic sclerostin loop3-LRP4 interaction required by sclerostin to impair whole-body lipid and glucose metabolism

doi: 10.1038/s41467-026-68526-w

Figure Lengend Snippet: a Experimental design. b Body weights of HFD-induced mice with or without Apc001OA treatment. c Serum sclerostin levels in HFD-induced mice with or without Apc001OA treatment. d Serum DKK1 levels in HFD-induced mice with or without Apc001OA treatment. e Fat pad weights in HFD-induced mice with or without Apc001OA treatment. gWAT: gonadal white adipose tissue; iWAT: inguinal white adipose tissue; rpWAT: retroperitoneal white adipose tissue; iBAT: interscapular brown adipose tissue. f Food intake in HFD-induced mice with or without Apc001OA treatment. Two-way ANOVA with Sidak’s multiple comparisons test was conducted to evaluate intergroup variations. g Serum free fatty acids in HFD-induced mice with or without Apc001OA treatment. h Representative images of histological sections (left) and frequency distribution of adipocyte sizes (right) in gWAT and iWAT from HFD-induced mice with or without Apc001OA treatment. Scale bars, 200 μm. i Expression levels of genes associated with lipid anabolism ( Acaca , Serbp2 , Lpl , and Fasn ) and catabolism ( Acads , Cpt1a , and Acadvl ) in gWAT from HFD-induced mice with or without Apc001OA treatment detected by qPCR. j Fasting blood glucose in HFD-induced mice with or without Apc001OA treatment. k Glucose metabolism in HFD-induced mice with or without Apc001OA treatment. Glucose tolerance test (GTT) (left), insulin tolerance test (ITT) (middle), and area under the curve (AUC) analysis for GTT and ITT (right). l Expression levels of genes associated with glucose metabolism in gWAT from HFD-induced mice with or without Apc001OA treatment detected by qPCR. Note: n = 7 biologically independent samples per group. All data were expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 for intergroup comparison (blue * : HFD + Vehicle vs. HFD + Apc001OA; gray * : HFD + Vehicle vs. HFD + Semaglutide). Statistical significance was calculated using unpaired t -test. ns: no significance. All tests were two-sided. Mouse image was created in BioRender. Zhang, G. (2025) https://BioRender.com/17da8dl .

Article Snippet: As previously described by Kim et al., sclerostin overproduction was achieved by injecting adeno-associated viruses (AAV8-CMV) encoding mouse SOST (Vector Biolabs, AAV-272868) via the tail vein to 6-month-old male C57BL/6J mice for 6 weeks .

Techniques: Expressing, Comparison

Journal: Nature Communications

Article Title: Adipocytic sclerostin loop3-LRP4 interaction required by sclerostin to impair whole-body lipid and glucose metabolism

doi: 10.1038/s41467-026-68526-w

Figure Lengend Snippet: a Binding ability of sclerostin to LRP4 by pull-down assay. b BLI analysis of the binding affinity between sclerostin and LRP4. ( c ) BLI analysis of the binding affinity between sclerostin loop3 and LRP4. d BLI analysis of the binding affinity between sclerostin and LRP4 in the presence of Apc001. e Binding ability of LRP4 muteins (LRP4 m46, m47, m67, m467) to sclerostin loop3 by pull-down assay for identifying the binding residues on LRP4 to sclerostin loop3. f TOP-Wnt luciferase signaling in 3T3-L1 cells with expression of LRP4 or LRP4 mutein in vitro in the presence of sclerostin. g Lipid droplet formation staining (upper) and quantification (lower) in 3T3-L1 cells with expression of LRP4 or LRP4 mutein in vitro in the presence of sclerostin. Scale bars, 100 μm. h qPCR analysis of lipid anabolism markers in 3T3-L1 cells with expression of LRP4 or LRP4 mutein in vitro in the presence of sclerostin. ( i ) qPCR analysis of lipid catabolism markers in 3T3-L1 cells with expression of LRP4 or LRP4 mutein in vitro in the presence of sclerostin. ( j ) qPCR analysis of glucose metabolism markers in 3T3-L1 cells with expression of LRP4 or LRP4 mutein in vitro in the presence of sclerostin. k Glucose uptake in 3T3-L1 cells with expression of LRP4 or LRP4 mutein in vitro in the presence of sclerostin. l Insulin-stimulated glucose uptake in 3T3-L1 cells with expression of LRP4 or LRP4 mutein in vitro in the presence of sclerostin. Note: 3T3-L1 cells: pre-adipocytes. PD: pull-down. n = 3 biologically independent samples. All data were expressed as mean ± SD. Statistical significance was calculated using unpaired t -test. ns no significance. All tests were two-sided.

Article Snippet: As previously described by Kim et al., sclerostin overproduction was achieved by injecting adeno-associated viruses (AAV8-CMV) encoding mouse SOST (Vector Biolabs, AAV-272868) via the tail vein to 6-month-old male C57BL/6J mice for 6 weeks .

Techniques: Binding Assay, Pull Down Assay, Luciferase, Expressing, In Vitro, Staining

Journal: Nature Communications

Article Title: Adipocytic sclerostin loop3-LRP4 interaction required by sclerostin to impair whole-body lipid and glucose metabolism

doi: 10.1038/s41467-026-68526-w

Figure Lengend Snippet: a TOP-Wnt luciferase signaling in LRP4-overexpressing 3T3-L1 cells with LA5 treatment in vitro in the presence of sclerostin. b Lipid droplet formation staining (left) and quantification (right) in LRP4-overexpressing 3T3-L1 cells with LA5 treatment in vitro in the presence of sclerostin. Scale bars, 100 μm. ( c ) qPCR analysis of lipid anabolism markers in LRP4-overexpressing 3T3-L1 cells with LA5 treatment in vitro in the presence of sclerostin. d qPCR analysis of lipid catabolism markers in LRP4-overexpressing 3T3-L1 cells with LA5 treatment in vitro in the presence of sclerostin. e qPCR analysis of glucose metabolism markers in LRP4-overexpressing 3T3-L1 cells with LA5 treatment in vitro in the presence of sclerostin. f Glucose uptake in LRP4-overexpressing 3T3-L1 cells with LA5 treatment in vitro in the presence of sclerostin. g Insulin-stimulated glucose uptake in LRP4-overexpressing 3T3-L1 cells with LA5 treatment in vitro in the presence of sclerostin. Note: 3T3-L1 cells: pre-adipocytes. n = 3 biologically independent samples. All data were expressed as mean ± SD. Statistical significance was calculated using unpaired t -test. ns no significance. All tests were two-sided.

Article Snippet: As previously described by Kim et al., sclerostin overproduction was achieved by injecting adeno-associated viruses (AAV8-CMV) encoding mouse SOST (Vector Biolabs, AAV-272868) via the tail vein to 6-month-old male C57BL/6J mice for 6 weeks .

Techniques: Luciferase, In Vitro, Staining

Journal: Nature Communications

Article Title: Adipocytic sclerostin loop3-LRP4 interaction required by sclerostin to impair whole-body lipid and glucose metabolism

doi: 10.1038/s41467-026-68526-w

Figure Lengend Snippet: a The schematic diagram of construction strategy for mouse models. b Body weights of Lrp4m/SOST ko mice and SOST ko mice with or without sclerostin overproduction. c Fat pad weights in Lrp4m/SOST ko mice and SOST ko mice with or without sclerostin overproduction. gWAT: gonadal white adipose tissue; iWAT: inguinal white adipose tissue; rpWAT: retroperitoneal white adipose tissue; iBAT: interscapular brown adipose tissue. d Serum free fatty acids in Lrp4m/SOST ko mice and SOST ko mice with or without sclerostin overproduction. e Representative images of histological sections (upper) and frequency distribution of adipocyte sizes (lower) in gWAT and iWAT from Lrp4m/SOST ko mice and SOST ko mice with or without sclerostin overproduction. Scale bars, 100 μm. f Expression levels of genes associated with lipid anabolism ( Acaca , Srebp2 , Lpl , and Fasn ) and catabolism ( Acads , Cpt1a , and Acadvl ) in gWAT from Lrp4m/SOST ko mice and SOST ko mice with or without sclerostin overproduction detected by qPCR. g Fasting blood glucose in Lrp4m/SOST ko mice and SOST ko mice with or without sclerostin overproduction. h Glucose metabolism in Lrp4m/SOST ko mice and SOST ko mice with or without sclerostin overproduction. Glucose tolerance test (GTT) (left), insulin tolerance test (ITT) (middle), and area under the curve (AUC) analysis for GTT and ITT (right). i Expression levels of genes associated with glucose metabolism in gWAT from Lrp4m/SOST ko mice and SOST ko mice with or without sclerostin overproduction detected by qPCR. Note: n = 6 biologically independent samples per group. All data were expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for intergroup comparison ( SOST ko vs. SOST ko + SOST). Statistical significance was calculated using unpaired t -test. ns: no significance. All tests were two-sided.

Article Snippet: As previously described by Kim et al., sclerostin overproduction was achieved by injecting adeno-associated viruses (AAV8-CMV) encoding mouse SOST (Vector Biolabs, AAV-272868) via the tail vein to 6-month-old male C57BL/6J mice for 6 weeks .

Techniques: Expressing, Comparison

Journal: Nature Communications

Article Title: Adipocytic sclerostin loop3-LRP4 interaction required by sclerostin to impair whole-body lipid and glucose metabolism

doi: 10.1038/s41467-026-68526-w

Figure Lengend Snippet: a Experimental design. b Body weights of SOST ki mice with or without LA5 treatment. c Fat pad weights in SOST ki mice with or without LA5 treatment. gWAT: gonadal white adipose tissue; iWAT: inguinal white adipose tissue; rpWAT: retroperitoneal white adipose tissue; iBAT: interscapular brown adipose tissue. d Serum free fatty acids in SOST ki mice with or without LA5 treatment. e Representative images of histological sections (left) and frequency distribution of adipocyte sizes (right) in gWAT and iWAT from SOST ki mice with or without LA5 treatment. Scale bars, 100 μm. f Expression levels of genes associated with lipid anabolism ( Acaca , Srebp2 , Lpl , and Fasn ) and catabolism ( Acads , Cpt1a , and Acadvl ) in gWAT from SOST ki mice with or without LA5 treatment detected by qPCR. g Fasting blood glucose in SOST ki mice with or without LA5 treatment. h Glucose metabolism in SOST ki mice with or without LA5 treatment. Glucose tolerance test (GTT) (left), insulin tolerance test (ITT) (middle), and area under the curve (AUC) analysis for GTT and ITT (right). i Expression levels of genes associated with glucose metabolism in gWAT from SOST ki mice with or without LA5 treatment detected by qPCR. Note: n = 6 biologically independent samples per group. All data were expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for intergroup comparison. Statistical significance was calculated using unpaired t -test. ns: no significance. All tests were two-sided. Mouse image was created in BioRender. Zhang, G. (2025) https://BioRender.com/17da8dl .

Article Snippet: As previously described by Kim et al., sclerostin overproduction was achieved by injecting adeno-associated viruses (AAV8-CMV) encoding mouse SOST (Vector Biolabs, AAV-272868) via the tail vein to 6-month-old male C57BL/6J mice for 6 weeks .

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B physically interacts with GTP-bound mutant KRAS and SOS. (a) Mass spectrometry analysis of eIF2B and KRAS interactions. (Left panel) eIF2B subunits specifically interact with the G12V mutant of FLAG-KRAS 4B, but not with the G12V mutant of FLAG-KRAS 4A. (Right panel) Mutations in the C-terminus hypervariable region of FLAG-KRAS G12V impair its interaction with the eIF2B subunits. V12, G12V mutation of KRAS 4B; CS, C→S mutation in the C-terminal CAAX motif of KRAS G12V; KQ, K→Q mutations in the poly-lysine stretch of KRAS G12V; Ctrl, mass spectrometry analysis of proteins bound to a non-target antibody. ( b , c ) HEK293T cells were co-transfected with MYC-tagged constructs for eIF2Bε (panel b) or all eIF2B subunits (panel c), together with KRAS G12V variants containing C→S and polyK→polyQ mutations within the HVR (panel b) as well as various KRAS mutants harboring substitutions at G12, G13, or Q61 (panel c). Cell lysates were subjected to IP with an anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-MYC antibodies to detect KRAS and eIF2B, respectively. Lysates from HEK293T cells transfected with insert less vector DNA served as negative controls. Protein loading in the co-IP assays was verified by immunoblotting of whole-cell extracts (WCE) ( d ) Interaction between endogenous eIF2B, SOS and KRAS in H358 and H1703 cells in co-IP assay with antibodies against the eIF2Bε subunit. Proteins were analyzed by immunoblotting to detect endogenous KRAS, SOS and eIF2Bε. Rabbit IgG was used as a negative control. Whole-cell extracts (WCE; 50 µg protein) were included as loading controls. ( e , f ) Cell lysates from H358 and H1703 cells were subjected to pull-down assays with GST-RBD of RAF. In panel (e), lysates from SOS1/2-proficient and SOS1/2 KD H358 cells were analyzed for bound KRAS, eIF2Bε, and SOS1 by immunoblotting. In panel (f), lysates from eIF2Bε-proficient and eIF2Bε-KD H358 (KRAS G12C) and H1703 (wild-type KRAS) cells were processed similarly using GST-RBD to detect KRAS, eIF2Bε, and SOS1. Whole-cell extracts (WCE; 50 µg protein) were included as loading controls.

Article Snippet: To assess ternary complex formation, 2 μg each of HIS-tagged human KRAS G12V (Acro Biosystems, Cat# KRS-H5143), Biotin-FLAG–tagged truncated SOS1 (BPS Bioscience, Cat# 100753), and recombinant tetrameric eIF2B (eIF2Bβγδε; ( )) were combined in 500 μL Lysis Buffer A. ISRIB was treated directly to a final concentration of 5 μM, with an equivalent volume of DMSO used as a negative control.

Techniques: Mutagenesis, Mass Spectrometry, Transfection, Construct, Western Blot, Plasmid Preparation, Co-Immunoprecipitation Assay, Negative Control

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: In silico assembly and biochemical mapping of the eIF2B:SOS:KRAS complex. (a) Putative model of the eIF2B:SOS:KRAS G12V structural assembly. eIF2B is predicted to associate either to the allosteric binding site of SOS or to GTP-bound RAS (dimer or oligomer) via its eIF2Bε subunit. In the close-up views of the predicted eIF2Bε interaction sites, the location of the mutated residues utilized in this study are highlighted with spheres on their Cα-atoms (eIF2Bε: blue spheres; SOS: orange spheres). For further information on the model see Suppl Figure 3. (b) The eIF2Bε subunit mediates the interaction of eIF2B with SOS and mutant KRAS. HEK293T cells were co-transfected with MYC-tagged constructs for each eIF2B subunit separately, HA-SOS1 and FLAG-KRAS G12V. Cell lysates were subjected to IP with an anti-MYC antibody, followed by immunoblotting with anti-FLAG, anti-HA, and anti-MYC antibodies to detect KRAS, SOS1, and eIF2B subunits, respectively. Protein loading in the co-IP assays was verified by immunoblotting of whole-cell extracts (WCE). (c) SOS residues 566–1046 (SOS CAT ) interacts with eIF2Bε and mutant KRAS, but to a lesser extent wild type (WT) KRAS. HEK293T cells were transfected with MYC-tagged eIF2Bε, T7-tagged SOS CAT , and either FLAG-tagged KRAS G12V or WT KRAS. Cell lysates were co-IPed using anti-MYC or anti-FLAG antibodies, followed by immunoblotting with anti-MYC, anti-T7, and anti-FLAG antibodies. (d) Mutations in the allosteric RAS-binding site of SOS CAT impair the interaction with eIF2Bε and mutant KRAS. HEK293T cells were transfected with MYC-tagged eIF2Bε, FLAG-tagged KRAS G12V and T7-tagged SOS CAT either wild type or containing the W729E or L687E/R688A mutations. Cell lysates were co-IPed with anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-T7, and anti-FLAG antibodies to detect the respective proteins. (e) The catalytic GEF activity of eIF2Bε is essential for its interaction with SOS and mutant KRAS. HEK293T cells were transfected with HA-SOS1, FLAG-KRAS G12V and MYC-eIF2Bε either wild-type or carrying the hyperactive D154A mutation, the catalytically inactive N263K mutation, or the QVA→ISP mutation in the C-terminus. Cell lysates were co-IPed with anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-HA, and anti-FLAG antibodies to detect the respective proteins. (f) Mutations in eIF2Bε impair its interaction with SOS and mutant KRAS. HEK293T cells were transfected with FLAG-tagged KRAS G12V, HA-tagged SOS1, and MYC-tagged eIF2Bε, either wild-type or containing the K103E, K141E, or K103E/K141E mutations. Cell lysates were subjected to co-IP with anti-FLAG or anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-HA, and anti-FLAG antibodies to detect the respective proteins. ( b - f ) Data represent one of three reproducible experiments.

Article Snippet: To assess ternary complex formation, 2 μg each of HIS-tagged human KRAS G12V (Acro Biosystems, Cat# KRS-H5143), Biotin-FLAG–tagged truncated SOS1 (BPS Bioscience, Cat# 100753), and recombinant tetrameric eIF2B (eIF2Bβγδε; ( )) were combined in 500 μL Lysis Buffer A. ISRIB was treated directly to a final concentration of 5 μM, with an equivalent volume of DMSO used as a negative control.

Techniques: In Silico, Binding Assay, Mutagenesis, Transfection, Construct, Western Blot, Co-Immunoprecipitation Assay, Activity Assay

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2Bε co-localizes with SOS and mutant KRAS at the PM. (a) IF analysis of MYC-eIF2Bε (red, 568 nm), HA-SOS1 (blue) and FLAG-KRAS (green), either WT (left) or G12V (right), co-expressed in HEK293T cells. MYC-tagged eIF2Bε was expressed in HEK293T cells either as wild-type or in mutant forms, including the hyperactive D154A mutant, the catalytically inactive N263K mutant, or the QVA→ISP mutant that disrupts interaction with SOS. Protein localization was assessed by confocal microscopy. The white signal in the merged images indicates the co-localization of the three proteins at the periphery of the cell. (b) IF analysis of cells described in panel (a) assessing PM localization of FLAG-tagged KRAS (green) following co-staining with Cholera Toxin subunit B (CT-B, far-red, 647 nm), a PM marker. Mander’s colocalization coefficients, analyzed with JACoP (ImageJ), were used to quantify the extent of FLAG-KRAS colocalization with CT-B at the PM. Scale bar: 5 μm. The yellow signal in the images indicates the co-localization of FLAG-KRAS and CT-B at the PM. (c) H358 cells (KRAS G12C) and H1703 cells (wild-type KRAS) expressing either scrambled shRNA or eIF2Bε shRNA were fractionated into nuclear (N), cytosolic (C), organelle (O), and plasma membrane (PM) fractions. Each fraction was subjected to immunoblotting to detect SOS1, eIF2Bε, and KRAS. Fractionation quality was verified using specific markers: EGFR for the PM, THOC1 for the nucleus, and α-TUBULIN for the cytosol.

Article Snippet: To assess ternary complex formation, 2 μg each of HIS-tagged human KRAS G12V (Acro Biosystems, Cat# KRS-H5143), Biotin-FLAG–tagged truncated SOS1 (BPS Bioscience, Cat# 100753), and recombinant tetrameric eIF2B (eIF2Bβγδε; ( )) were combined in 500 μL Lysis Buffer A. ISRIB was treated directly to a final concentration of 5 μM, with an equivalent volume of DMSO used as a negative control.

Techniques: Mutagenesis, Confocal Microscopy, IF-cells, Staining, Marker, Expressing, shRNA, Clinical Proteomics, Membrane, Western Blot, Fractionation

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B supports mutant KRAS PM localization and nanoclustering via the GSL pathway. (a) eIF2B specifically promotes mutant KRAS localization at PM. Representative confocal images of T47D cells expressing either GFP-KRAS G12V or GFP-HRAS G12V, treated with either scrambled shRNA or eIF2Bε shRNA. Cells were stained with CellMask to label the PM. Co-localization of GFP-KRAS with CellMask was quantified using Manders’ coefficient and is presented as mean ± SEM (n = 3). Scale bar: 10 μm (b) eIF2B controls the localization and spatial organization of mutant KRAS at the PM. PM sheets were isolated from H1703 cells stably expressing GFP-KRAS G12C and transfected with either scrambled or eIF2Bε siRNA. The PM sheets were labeled with anti-GFP-conjugated gold particles and visualized by EM. Representative EM images are shown. Quantification of gold particles is presented as mean number ± SEM (n = 32). Spatial distribution was analyzed, and L max values, indicating the extent of KRAS G12C clustering, are shown in bar graphs (n = 32). Statistical significance was assessed using Student’s t-test for gold particle count (left) and bootstrap test for L max (right). Numeric values indicate P -values. Scale bar: 0.1 μm. (c) eIF2Bε depletion reduces mutant KRAS clustering. PM sheets were isolated from T47D cells stably expressing GFP-KRAS G12V or GFP-HRAS G12V along with eIF2Bε shRNA. The PM sheets were labeled with anti-GFP-conjugated gold particles and visualized via EM. The number of gold particles is presented as mean ± SEM (n = 10). Spatial mapping was also performed, and peak L max values, reflecting the degree of protein clustering, are shown as bar graphs. Numeric values indicate P-values. ( d , e ) eIF2Bε KD significantly reduces the PM levels of GM3 and SM4. PM sheets from H358 cells (KRAS G12C; panel d) or Caco-2 cells overexpressing GFP-KRAS G12V (panel e), treated with either scrambled shRNA or eIF2Bε-targeting shRNA, were fixed and labeled with 4.5 nm gold-conjugated anti-GM3 or anti-SM4 antibodies, then imaged by EM. Spatial distribution of gold particles was analyzed using univariate K -functions (L(r) – r) . PM levels of GM3 and SM4 were quantified as gold particle density per 1 μm², and clustering was assessed by the peak value of L(r) – r ( L max ). Statistical significance for labeling density and L max was determined using Student’s t-test and bootstrap analysis, respectively (n ≥ 12, mean ± SEM). ( f ) Silencing of B4GALT5 specifically reduces GTP-bound KRAS in mutant KRAS-expressing cells. H358 (KRAS G12C) and H1703 (WT KRAS) cells were transfected with either scrambled control or B4GALT5 siRNA. Protein extracts were subjected to pull-down assays using GST–RBD of RAF, followed by immunoblotting with antibodies against KRAS, HRAS, eIF2Bε, SOS1, and B4GALT5. Protein loading was assessed by immunoblotting of whole-cell extracts (WCE).

Article Snippet: To assess ternary complex formation, 2 μg each of HIS-tagged human KRAS G12V (Acro Biosystems, Cat# KRS-H5143), Biotin-FLAG–tagged truncated SOS1 (BPS Bioscience, Cat# 100753), and recombinant tetrameric eIF2B (eIF2Bβγδε; ( )) were combined in 500 μL Lysis Buffer A. ISRIB was treated directly to a final concentration of 5 μM, with an equivalent volume of DMSO used as a negative control.

Techniques: Mutagenesis, Expressing, shRNA, Staining, Isolation, Stable Transfection, Transfection, Labeling, Control, Western Blot