Journal: International Journal of Oncology

Article Title: Carbon ion irradiation of the human prostate cancer cell line PC3: A whole genome microarray study

doi: 10.3892/ijo.2014.2287

Figure Lengend Snippet: The Applied Biosystems assays.

Article Snippet: ROCK1 , Rho-associated; coiled-coil containing protein kinase 1 , Hs01127714_mH , NM_005406.2 , 4–5 , 1.99.

Techniques:

Journal: International Journal of Oncology

Article Title: Carbon ion irradiation of the human prostate cancer cell line PC3: A whole genome microarray study

doi: 10.3892/ijo.2014.2287

Figure Lengend Snippet: Significantly regulated motility genes: FC ≤-2 or FC ≥2 and FDR ≤0.05.

Article Snippet: ROCK1 , Rho-associated; coiled-coil containing protein kinase 1 , Hs01127714_mH , NM_005406.2 , 4–5 , 1.99.

Techniques:

Journal: International Journal of Oncology

Article Title: Carbon ion irradiation of the human prostate cancer cell line PC3: A whole genome microarray study

doi: 10.3892/ijo.2014.2287

Figure Lengend Snippet: Microarray data of motility genes selected for RT-qPCR confirmation.

Article Snippet: ROCK1 , Rho-associated; coiled-coil containing protein kinase 1 , Hs01127714_mH , NM_005406.2 , 4–5 , 1.99.

Techniques: Microarray

Journal: International Journal of Oncology

Article Title: Carbon ion irradiation of the human prostate cancer cell line PC3: A whole genome microarray study

doi: 10.3892/ijo.2014.2287

Figure Lengend Snippet: Relative gene expression changes of six selected motility genes 8 h after carbon ion or X-irradiation. Log 2 (ratio) of the expression of (A) MYH9 , s(B) ROCK1 , (C) NEXN , (D) FN1 , (E) MYH10 and (F) CCDC88A is presented. Significantly altered gene expression compared to CTRL samples ( * P≤0.05) based on one-tailed Mann-Whitney tests. RT-qPCR results confirm the downregulation observed by microarray analysis after radiation which was more pronounced after carbon ion radiation when compared to X-rays.

Article Snippet: ROCK1 , Rho-associated; coiled-coil containing protein kinase 1 , Hs01127714_mH , NM_005406.2 , 4–5 , 1.99.

Techniques: Gene Expression, Irradiation, Expressing, One-tailed Test, MANN-WHITNEY, Quantitative RT-PCR, Microarray

Journal: International Journal of Oncology

Article Title: Carbon ion irradiation of the human prostate cancer cell line PC3: A whole genome microarray study

doi: 10.3892/ijo.2014.2287

Figure Lengend Snippet: Kaplan-Meier survival analysis of (A) CCDC88A , (B) FN1 , (C) MYH9 , (D) MYH10 , (E) NEXN and (F) ROCK1 gene expression performed on the data set of Taylor et al . Tumor samples were divided into three groups based on whether the gene expression value was high (♦, dark grey); intermediate (▴, light grey); or low (•, black). Differences in survival were found to be significant for CCDC88A , FN1 , NEXN and ROCK1 when log-rank P≤0.05.

Article Snippet: ROCK1 , Rho-associated; coiled-coil containing protein kinase 1 , Hs01127714_mH , NM_005406.2 , 4–5 , 1.99.

Techniques: Gene Expression

Journal: International Journal of Oncology

Article Title: Carbon ion irradiation of the human prostate cancer cell line PC3: A whole genome microarray study

doi: 10.3892/ijo.2014.2287

Figure Lengend Snippet: Kaplan-Meier survival analysis of (A) CCDC88A , (B) FN1 , (C) MYH9 , (D) MYH10 , (E) NEXN and (F) ROCK1 gene expression performed on the data set of Gulzar et al . Tumor samples were divided into three groups based on whether the gene expression value was high (♦, dark grey); intermediate (▴, light grey); or low (•, black). Differences in survival were found to be significant for FN1 (log-rank P=0.0257).

Article Snippet: ROCK1 , Rho-associated; coiled-coil containing protein kinase 1 , Hs01127714_mH , NM_005406.2 , 4–5 , 1.99.

Techniques: Gene Expression

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific protease 7-mediated stabilization of discoidin domain receptor 1 drives progression of TP53-Mutant cancers

doi: 10.1016/j.jbc.2025.110515

Figure Lengend Snippet: Mass-spectrum and DUBome identified USP7 as a stabilizing DUB for DDR1. A , endogenous DDR1 was immunoprecipitated from A549 cells, followed by mass spectrometry analysis. A DUBome screen of NSC632839 identified potential targets, including USP1, USP7, USP11, USP46, and USP47. USP7 was identified as a common target through the intersection of results from both methods. B , co-transfection of potential DUB targets and DDR1 in HEK293 T cells was performed to assess the stabilization effect on DDR1. DUBs were detected using either HA- or FLAG-tag-specific antibodies. C , HEK-293T cells were transfected with DDR1-Flag (0.5 μg) and Flag/HA-USP7 (0.5, 1, and 1.5 μg). DDR1 and HA-USP7 expression were detected by immunoblotting using the indicated antibodies. D , co-Immunoprecipitation (Co-IP) was performed with Myc IgG beads or DDR1 antibody in HEK-293T cells overexpressing DDR1-Flag and Myc-USP7. DDR1 and Myc-USP7 were detected in both IP and cell lysate (INPUT) by immunoblotting. E , reciprocal-co-IP was performed in A549 cells using DDR1 or USP7 antibodies, with IgG as a negative control. DDR1 and USP7 were detected in both IP and cell lysate (INPUT) by immunoblotting. Shown are the representative results of 3 independent experiments.

Article Snippet: The recombinant human-derived USP7 protein (purchased from MedChemExpress) was tested for activity in the Ubiquitin-AMC assay in the presence or absence of inhibitors.

Techniques: Immunoprecipitation, Mass Spectrometry, Cotransfection, FLAG-tag, Transfection, Expressing, Western Blot, Co-Immunoprecipitation Assay, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific protease 7-mediated stabilization of discoidin domain receptor 1 drives progression of TP53-Mutant cancers

doi: 10.1016/j.jbc.2025.110515

Figure Lengend Snippet: USP7 deubiquitinates and stabilizes DDR1, thereby promoting tumor cell growth in vitro and in vivo . A , HEK-293T cells were transfected with Flag-tagged DDR1, Myc-tagged USP7, and His-tagged ubiquitin, and treated with MG132 (20 μM) for 4 h prior to collection. Ubiquitinated DDR1 was pulled down using His IgG beads for detection. Ubiquitinated DDR1, USP7, and β-Actin were assessed by western blotting with the indicated antibodies ( upper panel ). The levels of ubiquitinated DDR1 were quantified as shown ( lower panel ). Mean ± SD (n = 3); Unpaired t test. B and C , USP7 was knocked down (KD) in A549 cells using USP7-silencing puromycin-resistant shRNA-1 via lentiviral transduction, with a scrambled (SCR) shRNA serving as a non-targeting control. Protein levels of DDR1, USP7, and β-Actin were assessed by western blotting with the indicated antibodies. qPCR was used to measure the mRNA levels of USP7 and DDR1, normalized to GAPDH. Mean ± SD (n = 4); Unpaired t test. D , A549 SCR and USP7-KD cells were treated with NSC632839 at varying concentrations for 16 h. DDR1, USP7, and GAPDH protein levels were detected by western blotting with the indicated antibodies. E , USP7 gene knockdown was performed in A549 cells. DDR1 immunoprecipitation (IP) was carried out, and ubiquitin, DDR1, and β-Actin protein levels were detected in both the IP and cell lysate (INPUT) by western blotting with the indicated antibodies ( left panel ). The levels of ubiquitinated DDR1 were quantified as shown ( right panel ). Mean ± SD (n = 3); Unpaired t test. F , long-term proliferation effects: Cells were generated as described in (E), and the growth curves of A549-shUSP7 cells were compared to those of SCR control cells using the CTG assay. Mean ± SD; Two-way ANOVA. G , short-term cytotoxicity assays: Cells were generated as described in (E), and the immediate effects of NSC632839 on different cell groups were assessed using the CTG assay. H , nude mice were implanted subcutaneously with 5e6 A549 cells (n = 5/group), 12 days later, 20 mg/kg (QD) of NSC632839 were given intraperitoneally. I , mice were treated with vehicle or NSC632839 (20 mg/kg) and sacrificed on day 15 of treatment. Tumor size measurements of xenograft mice after vehicle and NSC632839 treatment. Mean ± SEM; Two-way ANOVA. J , effects of 15 days NSC632839 treatment on growth of xenograft model were determined. Mean ± SEM; Unpaired t test. Tumor growth inhibition (TGI) rates were thus determined for both groups using the formula: TGI=((MTVcontrol-MTVtreated)/MTVcontrol) × 100. Shown are the representative results of 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The recombinant human-derived USP7 protein (purchased from MedChemExpress) was tested for activity in the Ubiquitin-AMC assay in the presence or absence of inhibitors.

Techniques: In Vitro, In Vivo, Transfection, Ubiquitin Proteomics, Western Blot, shRNA, Transduction, Control, Knockdown, Immunoprecipitation, Generated, CTG Assay, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific protease 7-mediated stabilization of discoidin domain receptor 1 drives progression of TP53-Mutant cancers

doi: 10.1016/j.jbc.2025.110515

Figure Lengend Snippet: TP53 mutation or depletion promotes DDR1 expression and its association with USP7. A , DDR1 protein levels were quantitatively analyzed in DLBCL cell lines with TP53-WT and TP53-MUT. Mean ± SD; Unpaired t test. B , tissue microarray (TMA): Tumor tissue samples from 28 DLBCL patients were classified into 2 groups based on in situ FISH analysis: TP53-WT (n = 22) and TP53-deletion (n = 6). DDR1 expression was assessed by immunohistochemical staining, with staining intensity (scored from 0 to 3) and positivity rate (scored from 0 to 4) evaluated separately. The composite score, ranging from 0 to 12, was calculated as the product of these 2 scores. Scale bar represents 50 μm. C , statistical analysis corresponding to the data shown in panel (B) is presented in this panel. Mean ± SD; Unpaired t test. D , HEK-293T cells were co-transfected with a DDR1 promoter-driven firefly luciferase reporter containing either wild-type (WT) or mutated (MUT) binding sites, with or without TP53, and incubated for 48 h before harvesting for luciferase activity measurement. Mean ± SD (n = 6); One-way ANOVA. E and F , TP53 was knocked down in A549 cells using puromycin-resistant shTP53-1 and shTP53-2 lentiviral constructs, with scrambled (SCR) shRNA as a non-targeting control. DDR1, TP53, and β-Actin protein levels were evaluated by western blotting with the indicated antibodies. qPCR was used to measure the mRNA levels of DDR1 and TP53, normalized to GAPDH. Mean ± SD (n = 6); One-way ANOVA. G , A549 and Pfeiffer cells were treated with NSC632839 for 16 h at concentrations of 0, 2.5, 5, and 10 μM. DDR1, TP53, MMP2, β-Actin, and GAPDH protein levels were assessed by western blotting using the specified antibodies. H , HEK-293T SCR and TP53-KD cells were transfected with Flag-tagged DDR1 and Myc-tagged USP7. Myc pulldown was performed, and DDR1 and Myc protein levels were detected in both immunoprecipitations (IPs) and cell lysates (INPUT) by western blotting using the indicated antibodies. Shown are the representative results of 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The recombinant human-derived USP7 protein (purchased from MedChemExpress) was tested for activity in the Ubiquitin-AMC assay in the presence or absence of inhibitors.

Techniques: Mutagenesis, Expressing, Microarray, In Situ, Immunohistochemical staining, Staining, Transfection, Luciferase, Binding Assay, Incubation, Activity Assay, Construct, shRNA, Control, Western Blot

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: IQGAP1 binds directly to the MET receptor. ( A ). GST-IQGAP1 (IQ1), bound to glutathione-Sepharose beads, was incubated with 2 μg of the purified intracellular region of MET (residues 974–1390). Control pull-downs were carried out with GST-glutathione-Sepharose (GST). After washing, proteins attached to the beads were eluted in Laemmli sample buffer and analyzed by SDS-PAGE. The gel was cut at ∼ 80 kDa. The top portion of the gel was stained with Coomassie blue. The lower portion was processed by Western blotting and probed with anti-MET and anti-GST antibodies. Input designates pure MET not subjected to pull-down. The positions of migration of molecular weight markers are indicated on the left. All images are from the same gel. The data shown are representative of two independent experiments. The multiple bands observed on the MET blot likely reflect partial degradation. ( B ). H1993 cells were cultured and lysed. Equal amounts of protein from cell lysates were subjected to immunoprecipitation (IP) with an anti-MET antibody. Control precipitation was carried out with mouse IgG. Samples were resolved by SDS-PAGE followed by Western blotting and probed with anti-MET and anti-IQGAP1 (IQ1) antibodies. Unfractionated cell lysate (Input) was processed in parallel. Both images are from the same membrane. ( C ). Immunoprecipitation was carried out from H1993 cell lysates as described for ( B ), except anti-IQGAP1 antibody was used. Control precipitation was carried out with non-immune rabbit serum (NIRS). Western blots were probed with anti-IQGAP1 (IQ1) and anti-MET antibodies. Both images are from the same membrane. All blots in panels ( B , C ) are representative of three independent experiments. Data presented in panels ( A–C ) were cropped from the full immunoblots shown in . ( D ). H1993 cells grown on coverslips were fixed, permeabilized, and incubated with anti-MET and anti-IQGAP1 antibodies. A proximity ligation assay (PLA) was carried out using Duolink in situ probes and detection reagents (Millipore Sigma). Cell images were acquired via confocal microscopy. Red spots indicate positive PLA signals. Actin was stained with phalloidin (green). Scale bar, 10 μm. Two representative images from two independent experiments (80 cells each) are shown. ( E ). HepG2 cells were incubated in the absence (left panels) or presence (right panels) of HGF. A PLA was performed as described for ( D ), and foci were quantified from confocal images using ImageJ. The boxplot shows the distribution of the number of PLA dots per cell observed with or without HGF (horizontal line, median; box, 25–75% percentiles; whiskers, 10–90% percentiles; n = 100). Statistical analysis was carried out using an unpaired t -test (***, p ≤ 0.001).

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Incubation, Purification, Control, SDS Page, Staining, Western Blot, Migration, Molecular Weight, Cell Culture, Immunoprecipitation, Membrane, Proximity Ligation Assay, In Situ, Confocal Microscopy

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: Knockdown of IQGAP1 enhances MET activation and signaling. HepG2 cells were transfected with control siRNA (siCtrl) or IQGAP1 siRNA (siIQ1). Next, 48 h after transfection, cells were starved of serum for 16 h and then incubated with 50 ng/mL HGF for 0, 5, or 10 min. Equal amounts of cell lysates were analyzed by SDS-PAGE and Western blotting. ( A ). The membrane was probed with an anti-IQGAP1 antibody. An anti-tubulin antibody was used for the loading control. ( B ). The intensity of the IQGAP1 band was quantified using Image Studio 2.0 (LI-COR Biosciences) and corrected for that of tubulin in the corresponding sample. Data are expressed as the IQGAP1/tubulin ratio from the three HGF timepoints for three independent repetitions (mean ± SD, n = 9). The ratio observed for siCtrl cells was set as 1. ( C ). Antibodies specific to phosphorylated MET (pMET, Tyr 1234 /Tyr 1235 ) and total MET were used to probe the Western blot membrane. ( D ). The pMET bands were quantified and corrected for the amount of total MET in the corresponding sample. ( E ). The membrane was probed with antibodies to phosphorylated Akt (pAkt, Ser 473 ) and total Akt. ( F ). The pAkt and Akt signals were quantified and the pAkt/Akt ratio was calculated. ( G ). Phosphorylated ERK (pERK, Thr 202 /Tyr 204 ) and total ERK were detected on the Western blot membrane using specific antibodies. ( H ). The pERK bands were quantified and corrected for the amount of total ERK in the corresponding sample. Graphs D, F, and H show the means ± SD from three independent experiments. For these graphs, the ratio of phosphorylated to total protein in the absence of HGF (0 min) was set as 1 for both siCtrl and siIQ1 cells. Statistical analyses were performed using unpaired t -tests (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). All blots in this figure are representative of three independent experiments. The full blots of all three replicates are shown in .

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knockdown, Activation Assay, Transfection, Control, Incubation, SDS Page, Western Blot, Membrane

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: Phosphorylation of Tyr 1510 of IQGAP1 creates a docking site for SH2 domains. ( A ). A peptide array containing 124 different GST-tagged recombinant SH2 protein domains, each spotted in duplicate, was generated as detailed in Materials and Methods . GST alone was spotted on the array as the negative control. Biotinylated peptides comprising residues 1502–1518 of IQGAP1 with phosphorylated Tyr 1510 (pTyr 1510 ) were labeled with fluorescent streptavidin and incubated with the array for 16 h at 4 °C. After washing, fluorescence from the bound peptides (green dots) was detected. The red and orange circles delineate the duplicates for the SH2 domains of Abl1 and Abl2, respectively. ( B ). Similar analysis was conducted with the same IQGAP1 peptide, except Tyr 1510 was not phosphorylated. ( C ). The peptide array was probed with anti-GST antibody to show the positions and loading of the GST-SH2 domains and control GST.

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Phospho-proteomics, Peptide Microarray, Recombinant, Generated, Negative Control, Labeling, Incubation, Fluorescence, Control

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: The SH2 domains of Abl1 and Abl2 bind directly to tyrosine-phosphorylated IQGAP1. ( A ). Quantification of the fluorescence intensity of the IQGAP1 peptides with unphosphorylated (Tyr 1510 , pale green bars) or phosphorylated (pTyr 1510 , dark green bars) Tyr 1510 bound to the SH2 domains of Abl1 or Abl2 on the SH2 array. Binding to GST is the control. Data represent the mean fluorescence from two duplicates. a.u., arbitrary units. ( B ). H1993 cells were incubated with 100 nM crizotinib (criz, + ) or vehicle DMSO (−). After 24 h, cells were lysed and equal amounts of protein lysate were incubated with the purified GST-SH2 domains of Abl1 or Abl2 bound to glutathione-Sepharose. Control pull-downs were carried out with GST-glutathione-Sepharose. After washing, proteins attached to the beads were eluted in Laemmli sample buffer and analyzed by Western blotting. The membrane was probed with anti-IQGAP1 (IQ1) and anti-GST antibodies. Input designates unfractionated cell lysates. Both panels are from the same membrane. Blots are representative of two independent experiments. The full blots of the two replicates are shown in . ( C ). The IQGAP1 bands observed after pull-down were quantified using Image Studio 2.0 (LI-COR Biosciences). The intensity of IQGAP1 in DMSO-treated cells was set as 1. Data are presented as the means of two independent replicates. ( D ). Purified GST-IQGAP1 (IQ1) on glutathione-Sepharose was incubated with purified active MET in the presence (+ATP) or absence (−ATP) of ATP. After washing, beads were incubated with 2 μg of purified Abl1 (left panel) or Abl2 (right panel). Control pull-downs were carried out with GST-Sepharose. After washing, proteins attached to the beads were eluted in Laemmli sample buffer and analyzed by SDS-PAGE and Western blotting. The membrane was probed with anti-IQGAP1 (IQ1), anti-phosphotyrosine (pTyr), and anti-Abl1 or anti-Abl2 antibodies. The overlap between IQGAP1 (red) and pTyr (green) signals is visible in the merged image (yellow). Input designates pure Abl1 or Abl2 not subjected to pull-down. The blots are representative of three independent experiments. The full blots of the three replicates are shown in . ( E ). The Abl1 and Abl2 bands observed after pull-down by GST-IQGAP1 were quantified using Image Studio 2.0 (LI-COR Biosciences). The intensity of Abl1 and Abl2 signals observed with non-phosphorylated IQGAP1 (−ATP) was set as 1. Data are the means ± SD of three independent experiments. Statistical analyses were performed with unpaired t -tests (*, p ≤ 0.05).

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Fluorescence, Binding Assay, Control, Incubation, Purification, Western Blot, Membrane, SDS Page

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: Endogenous IQGAP1 with phosphorylated Tyr 1510 binds to endogenous Abl1 and Abl2. ( A ). H1993 cells were cultured and lysed. Equal amounts of protein from cell lysates were subjected to immunoprecipitation (IP) with anti-Abl1 (left panel) or anti-Abl2 (right panel) antibodies. Control precipitations were carried out with mouse IgG. Samples were resolved by SDS-PAGE followed by Western blotting and probed with antibodies to IQGAP1 (IQ1) and Abl1 or Abl2. Unfractionated cell lysates (Input) were processed in parallel. Both images in each panel are from the same membrane. Blots are representative of three independent experiments. The presented data were cropped from the full immunoblots shown in . ( B ). H1993 cells were incubated with 100 nM crizotinib (criz, +) or vehicle DMSO (−) for 24 h. Cells were then lysed, Abl1 or Abl2 was immunoprecipitated, and samples were resolved by Western blotting as described for panel A. The amount of IQGAP1 was quantified and corrected for the amount of immunoprecipitated Abl1 or Abl2 in the same sample. Ratios were set as 1 for DMSO-treated (−) cells. Data are the means ± SD of three independent repetitions. Statistical analyses were performed with unpaired t -tests (***, p ≤ 0.001). All blots shown in this figure are representative of three independent experiments. The full blots of the three replicates are shown in .

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, Immunoprecipitation, Control, SDS Page, Western Blot, Membrane, Incubation

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: IQGAP1 modulates Abl kinase activity. HepG2 cells were transfected with scrambled siRNA (siCtrl) or siIQGAP1 (siIQ1) RNA. After 48 h, cells were starved for 16 h before being incubated with 50 ng/mL HGF for 0, 5, or 10 min. ( A ). Cells were lysed and equal amounts of protein lysates were analyzed by Western blotting using anti-IQGAP1 and anti-tubulin antibodies. ( B ). IQGAP1 signal intensity was quantified with Image Studio 2.0 (LI-COR Biosciences) and corrected for the amount of tubulin in the corresponding sample. Data are expressed as the IQGAP1/tubulin ratio from the three HGF timepoints for four independent repetitions (mean ± SD, n = 12). The ratio in siCtrl cells was set as 1. ( C ). Western blots of the same HepG2 cell lysates as those used for panel A were probed for CrkL phosphorylated on Tyr 207 (pCrkL) and total CrkL. ( D ). The pCrkL band at the 0 min HGF timepoint (basal phosphorylation) was quantified and corrected for the amount of total CrkL in the corresponding sample. siCtrl cells were set at 1 (mean ± SD, n = 4). ( E ). The pCrkL/CrkL ratio was calculated for the 0 and 10 min HGF timepoints. The ratio for 0 min HGF was set at 1 for both siCtrl and siIQ1 cells to visualize the effect of HGF stimulation on pCrkL (mean ± SD, n = 4). ( F ). The CrkL band was quantified and corrected for the amount of tubulin in the same sample. Data are expressed as the IQGAP1/tubulin ratio from the three HGF timepoints for four independent repetitions (mean ± SD, n = 12). All blots shown in this figure are representative of four independent experiments. Statistical analyses were carried out using unpaired t -tests (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). The full blots of the four replicates are shown in .

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activity Assay, Transfection, Incubation, Western Blot, Phospho-proteomics

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: Model of IQGAP1 in receptor tyrosine kinase signaling. ( A ). Schematic of IQGAP1 that highlights known post-translational modifications. The five domains are CHD (calponin-homology domain), WW, IQ, GRD (GAP-related domain), and RGCT (RasGAP_C-terminus). Post-translational modifications that have been characterized on IQGAP1 are shown below the modified amino acid. The identified domain to which receptor tyrosine kinases bind is depicted above IQGAP1. Phosphorylation of IQGAP1 on Tyr 1510 , leading to the recruitment of SH2 domains, is also shown. ( B ). Model depicting the identified modes of action of IQGAP1 in receptor tyrosine kinase signaling. Upper panel: constitutive scaffolding by IQGAP1. IQGAP1 binds constitutively to both the growth factor receptor and a downstream signaling protein. (i) Ligand binding activates the receptor. (ii) Scaffolding by IQGAP1 facilitates activation of the signaling protein by the activated receptor, initiating downstream signaling. (iii) Signal transduction from the activated receptor to the effector protein does not occur in the absence of scaffolding by IQGAP1. The mode of action depicted here is for the EGF receptor (EGFR) and B-Raf kinase in activation of the MAPK cascade [ , ]. Lower panel: phosphotyrosine-dependent scaffolding by IQGAP1. (i) Phosphorylation of tyrosine on IQGAP1 by an activated receptor tyrosine kinase (ii) initiates recruitment of selected SH2-containing proteins. The mechanism illustrated here is for the MET receptor tyrosine kinase and the SH2-containing proteins Abl1 and Abl2. In this example, IQGAP1 (iii) impairs MET activation and signaling and (iv) decreases HGF-stimulated signaling of Abl to the adaptor protein CrkL. Therefore, IQGAP1 functions as a rheostat regulating the flux of signaling between activated MET receptors and SH2-containing signaling proteins. (v) Recruitment of SH2-containing proteins does not occur in the absence of receptor tyrosine kinase-catalyzed phosphorylation of IQGAP1. Abbreviations: EGF, epidermal growth factor; EGFR, EGF receptor; HER2, human epidermal growth factor receptor 2; HGF, hepatocyte growth factor; IR, insulin receptor; P, phosphate; pS, serine phosphorylation; pY, tyrosine phosphorylation; SUMO, SUMOylation; Ub, ubiquitination.

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Modification, Phospho-proteomics, Scaffolding, Ligand Binding Assay, Activation Assay, Transduction, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: Human BCL-G regulates secretion of inflammatory chemokines but is dispensable for induction of apoptosis by IFN-γ and TNF-α in intestinal epithelial cells

doi: 10.1038/s41419-020-2263-0

Figure Lengend Snippet: a Relative mRNA expression of BCL-G S/L measured by RT-qPCR in the indicated adult human tissues. Relative mRNA expression of b BCL-G S and c BCL-G L measured by RT-qPCR in colonic biopsy tissues isolated from non-IBD individuals ( n = 20), patients with inactive ( n = 20) or active ( n = 24) ulcerative colitis, and patients with inactive ( n = 19) or active ( n = 21) Crohn’s disease. BCL-G S/L expression data in active ulcerative colitis were used for correlation analysis in Fig. . For panels ( b ) and ( c ), data shown include the median with interquartile range. d Relative mRNA expression of BCL-G S/L measured by RT-qPCR in TissueScan cDNA array of 24 matched samples (normal, uninvolved colon vs. colorectal cancer) covering clinical stage I ( n = 5), II ( n = 7), III ( n = 8) and IV ( n = 4). # p < 0.05, ### p < 0.001 (Kruskal–Wallis test followed by Dunn’s multiple comparisons test as indicated), $ p < 0.05, $$$ p < 0.001 (repeated measures two-way ANOVA followed by Fisher’s LSD test as indicated), ‡‡ p < 0.01, ‡‡‡ p < 0.001 (repeated measures two-way ANOVA followed by Fisher’s LSD test vs. normal colon, stage I). IBD — inflammatory bowel disease, UC — ulcerative colitis, CD — Crohn’s disease.

Article Snippet: To study cancer-associated gene expression, Colon Cancer TissueScan cDNA array III (cat# HCRT303, OriGene) was used.

Techniques: Expressing, Quantitative RT-PCR, Isolation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 1 HN1 is overexpressed in human HCC and correlates with poor prognosis and promoter methylation. A Kaplan–Meier analysis for the overall survival times of hepatocellular carcinoma patients with high HN1 expression and low HN1 expression in TCGA (left) and the NCI (right) database. TCGA, The Cancer Genome Atlas. NCI, National Cancer Institute. OS, overall survival. B The correlation between HN1 mRNA expression and HN1 DNA methylation was analyzed in HCC patients. C The protein expression of HN1 in paired HCC tissues and adjacent non- tumoral liver tissues from seven patients. T liver tumor tissue. N adjacent non-tumoral liver tissue. D The protein expression of HN1 in unpaired HCC tissues from 7 patients. E The relative levels of HN1 mRNA were examined using qPCR in paired HCC and adjacent non-tumoral liver tissues from seven patients. F The protein levels of HN1 in seven hepatocellular carcinoma cell lines were detected. Data were expressed as the mean ± SEM of three independent experiments. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Methylation, Expressing, DNA Methylation Assay, Software, Control

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 2 Effects of HN1 on cell proliferation and apoptosis in HCC. A WST-1 assay to assess the viability of the HepG2 and SNU449 cell lines after HN1 shRNA knockdown or HN1 overexpression. B A colony staining assay showed the colony formation ability of HepG2 and SNU449 cells with HN1 shRNA knockdown or HN1 overexpression. Colonies were counted at least in five fields. C Apoptosis marker proteins (PARP/ cleaved-PARP and caspase-9/cleaved-caspase-9) were detected in pairs by western blots of HepG2 and SNU449 cells after HN1 shRNA knockdown or HN1 overexpression. GAPDH was used as the internal control. Data were expressed as the mean ± SEM of three independent experiments. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control. shCTRL, control shRNA (control). shHN1, HN1 shRNA knockdown. Vector, control vector plasmid (control). HN1 OE HN1 overexpression.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: WST-1 Assay, shRNA, Knockdown, Over Expression, Staining, Marker, Western Blot, Control, Software, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 3 Effects of HN1 on the cell cycle in HCC. A FACs cell cycle analysis of the HepG2 and SNU449 cell lines with HN1 shRNA knockdown. B Western blot analysis of the G1 cycle-related proteins CDK4, CDK6, Cyclin D1, and p53 in the HepG2 and SNU449 cell lines with HN1 shRNA knockdown. GAPDH was used as the internal control. Data represent the mean ± SEM of three independent experiments. *p < 0.05 and **p < 0.01 compared with the corresponding control. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Cell Cycle Assay, shRNA, Knockdown, Western Blot, Control

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 4 Effects of HN1 on cell migration and invasion in HCC. A Wound-healing assay to assess the cell migration rate in the HepG2 and SNU449 cell lines after HN1 shRNA knockdown or HN1 overexpression. Representative images were obtained at 0, 24, and 48 h. Migration ability was quantified by measuring the gap distance. B Matrigel transwell assay to assess the cell invasion ability of the HepG2 and SNU449 cell lines after HN1 shRNA knockdown or HN1 overexpression for 72 h. The migration-related molecular uPA and vimentin protein levels (C) and mRNA levels (D) were detected by western blotting and rt-qPCR, respectively, in HepG2 and SNU449 cells after HN1 shRNA knockdown or HN1 overexpression. GAPDH was used as the internal control. Data are expressed as the mean ± SEM of three independent experiments. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown, Vector control vector plasmid (control), HN1 OE HN1 overexpression.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Migration, Wound Healing Assay, shRNA, Knockdown, Over Expression, Transwell Assay, Western Blot, Quantitative RT-PCR, Control, Software, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 5 Gene expression levels affected by HN1 knockdown in HCC. A cDNA microarray heatmap showing the effects of HN1 shRNA knockdown on AKT signaling pathway-related genes in HCC cells. The data were expressed in matrix format, with rows representing individual genes and columns representing individual samples. Red and green indicate increased and decreased gene expression, respectively. B Gene ontology analysis showed that HN1 knockdown was related to a series of diseases and disorders. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Gene Expression, Knockdown, Microarray, shRNA, Control

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 7 HN1 regulated SREBP-1 and SREBP-2 in HCC. A Downstream gene networks from the ingenuity pathway analysis. HN1 knockdown inhibited the SREBF-1 and SREBF-2 genes and their downstream genes. B Precursor and mature SREBP-1c protein expression was examined by western blotting in HepG2 and SNU449 cells after HN1 shRNA knockdown or HN1 overexpression. GAPDH was used as the internal control. C SREBP-1 cell distribution was observed directly by immunofluorescence against the SREBP-1 antibody. Green, SREBP-1. Blue, DAPI (nucleus). The distribution of fluorescence density in the nucleus and protein quantification were measured by ImageJ software. D The mRNA levels of precursor and mature SREBP-1 were measured by rt-qPCR after HN1 shRNA knockdown in the HepG2 and SNU449 cell lines. Data were expressed as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control. shCTRL control shRNA (control). shHN1 HN1 shRNA knockdown, Vector control vector plasmid (control), HN1 OE HN1 overexpression.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Knockdown, Expressing, Western Blot, shRNA, Over Expression, Control, Software, Quantitative RT-PCR, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 10 Knockdown of HN1 inhibited tumorigenesis in xenograft mice. The xenograft nude mouse models were established with HepG2 cells transfected with control shRNA or HN1 shRNA. The mice were separated into two groups: (i) control shRNA group and (ii) HN1 shRNA group. A, B Images of mice and tumors. Body weight (C) and tumor volume (E) were measured every 3 days. Tumor weight (D) and images of tumor size (B) were evaluated after euthanasia. F A histopathological analysis of H&E stained tissues and the TUNEL assay were performed to determine the histological characteristics of the mouse tumor tissues. G Tumor tissues were immunostained with HN1 and SREBP-1 antibodies. H Western blot analysis of HN1, precursor and mature SREBP-1C, FAS, and ACC in mouse tumor tissues. GAPDH was used as the cytoplasm internal control. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Knockdown, Transfection, Control, shRNA, Staining, TUNEL Assay, Western Blot, Software

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 11 Inhibition of SREBP reverses the tumorigenic effect of HN1 in xenograft mice. Xenograft nude mouse models were established using HepG2 cells transfected with either a control or HN1 overexpression vector. The mice were divided into three groups: (i) control group, (ii) HN1 overexpression group, and (iii) HN1 overexpression combined with Fatostatin. A Tumor volume was measured every 3 days. After euthanasia, images of the tumors (B), body weight (C), and tumor weight (D) were evaluated. *p < 0.05, **p < 0.01, and ***p < 0.001: vector vs. HN1 OE. ## p < 0.01 and ###p < 0.001: HN1 OE vs. HN1 OE plus Fatostatin. “Vector” refers to the control vector plasmid, “HN1 OE” to HN1 overexpression, and “Fatostatin” to the SREBP inhibitor.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Inhibition, Transfection, Control, Over Expression, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 12 Schematic representation of HN1-mediated inhibition of tumorigenesis of HCC. HN1 suppression deactivates the Akt pathway, leading to reduced levels of mature SREBP in the nucleus, thereby inhibiting lipogenesis and ultimately suppressing hepato- cellular carcinoma (HCC) tumorigenesis.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Inhibition

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 1 HN1 is overexpressed in human HCC and correlates with poor prognosis and promoter methylation. A Kaplan–Meier analysis for the overall survival times of hepatocellular carcinoma patients with high HN1 expression and low HN1 expression in TCGA (left) and the NCI (right) database. TCGA, The Cancer Genome Atlas. NCI, National Cancer Institute. OS, overall survival. B The correlation between HN1 mRNA expression and HN1 DNA methylation was analyzed in HCC patients. C The protein expression of HN1 in paired HCC tissues and adjacent non- tumoral liver tissues from seven patients. T liver tumor tissue. N adjacent non-tumoral liver tissue. D The protein expression of HN1 in unpaired HCC tissues from 7 patients. E The relative levels of HN1 mRNA were examined using qPCR in paired HCC and adjacent non-tumoral liver tissues from seven patients. F The protein levels of HN1 in seven hepatocellular carcinoma cell lines were detected. Data were expressed as the mean ± SEM of three independent experiments. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Methylation, Expressing, DNA Methylation Assay, Software, Control

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 2 Effects of HN1 on cell proliferation and apoptosis in HCC. A WST-1 assay to assess the viability of the HepG2 and SNU449 cell lines after HN1 shRNA knockdown or HN1 overexpression. B A colony staining assay showed the colony formation ability of HepG2 and SNU449 cells with HN1 shRNA knockdown or HN1 overexpression. Colonies were counted at least in five fields. C Apoptosis marker proteins (PARP/ cleaved-PARP and caspase-9/cleaved-caspase-9) were detected in pairs by western blots of HepG2 and SNU449 cells after HN1 shRNA knockdown or HN1 overexpression. GAPDH was used as the internal control. Data were expressed as the mean ± SEM of three independent experiments. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control. shCTRL, control shRNA (control). shHN1, HN1 shRNA knockdown. Vector, control vector plasmid (control). HN1 OE HN1 overexpression.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: WST-1 Assay, shRNA, Knockdown, Over Expression, Staining, Marker, Western Blot, Control, Software, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 3 Effects of HN1 on the cell cycle in HCC. A FACs cell cycle analysis of the HepG2 and SNU449 cell lines with HN1 shRNA knockdown. B Western blot analysis of the G1 cycle-related proteins CDK4, CDK6, Cyclin D1, and p53 in the HepG2 and SNU449 cell lines with HN1 shRNA knockdown. GAPDH was used as the internal control. Data represent the mean ± SEM of three independent experiments. *p < 0.05 and **p < 0.01 compared with the corresponding control. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Cell Cycle Assay, shRNA, Knockdown, Western Blot, Control

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 4 Effects of HN1 on cell migration and invasion in HCC. A Wound-healing assay to assess the cell migration rate in the HepG2 and SNU449 cell lines after HN1 shRNA knockdown or HN1 overexpression. Representative images were obtained at 0, 24, and 48 h. Migration ability was quantified by measuring the gap distance. B Matrigel transwell assay to assess the cell invasion ability of the HepG2 and SNU449 cell lines after HN1 shRNA knockdown or HN1 overexpression for 72 h. The migration-related molecular uPA and vimentin protein levels (C) and mRNA levels (D) were detected by western blotting and rt-qPCR, respectively, in HepG2 and SNU449 cells after HN1 shRNA knockdown or HN1 overexpression. GAPDH was used as the internal control. Data are expressed as the mean ± SEM of three independent experiments. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown, Vector control vector plasmid (control), HN1 OE HN1 overexpression.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Migration, Wound Healing Assay, shRNA, Knockdown, Over Expression, Transwell Assay, Western Blot, Quantitative RT-PCR, Control, Software, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 5 Gene expression levels affected by HN1 knockdown in HCC. A cDNA microarray heatmap showing the effects of HN1 shRNA knockdown on AKT signaling pathway-related genes in HCC cells. The data were expressed in matrix format, with rows representing individual genes and columns representing individual samples. Red and green indicate increased and decreased gene expression, respectively. B Gene ontology analysis showed that HN1 knockdown was related to a series of diseases and disorders. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Gene Expression, Knockdown, Microarray, shRNA, Control

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 7 HN1 regulated SREBP-1 and SREBP-2 in HCC. A Downstream gene networks from the ingenuity pathway analysis. HN1 knockdown inhibited the SREBF-1 and SREBF-2 genes and their downstream genes. B Precursor and mature SREBP-1c protein expression was examined by western blotting in HepG2 and SNU449 cells after HN1 shRNA knockdown or HN1 overexpression. GAPDH was used as the internal control. C SREBP-1 cell distribution was observed directly by immunofluorescence against the SREBP-1 antibody. Green, SREBP-1. Blue, DAPI (nucleus). The distribution of fluorescence density in the nucleus and protein quantification were measured by ImageJ software. D The mRNA levels of precursor and mature SREBP-1 were measured by rt-qPCR after HN1 shRNA knockdown in the HepG2 and SNU449 cell lines. Data were expressed as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control. shCTRL control shRNA (control). shHN1 HN1 shRNA knockdown, Vector control vector plasmid (control), HN1 OE HN1 overexpression.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Knockdown, Expressing, Western Blot, shRNA, Over Expression, Control, Software, Quantitative RT-PCR, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 10 Knockdown of HN1 inhibited tumorigenesis in xenograft mice. The xenograft nude mouse models were established with HepG2 cells transfected with control shRNA or HN1 shRNA. The mice were separated into two groups: (i) control shRNA group and (ii) HN1 shRNA group. A, B Images of mice and tumors. Body weight (C) and tumor volume (E) were measured every 3 days. Tumor weight (D) and images of tumor size (B) were evaluated after euthanasia. F A histopathological analysis of H&E stained tissues and the TUNEL assay were performed to determine the histological characteristics of the mouse tumor tissues. G Tumor tissues were immunostained with HN1 and SREBP-1 antibodies. H Western blot analysis of HN1, precursor and mature SREBP-1C, FAS, and ACC in mouse tumor tissues. GAPDH was used as the cytoplasm internal control. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Knockdown, Transfection, Control, shRNA, Staining, TUNEL Assay, Western Blot, Software

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 11 Inhibition of SREBP reverses the tumorigenic effect of HN1 in xenograft mice. Xenograft nude mouse models were established using HepG2 cells transfected with either a control or HN1 overexpression vector. The mice were divided into three groups: (i) control group, (ii) HN1 overexpression group, and (iii) HN1 overexpression combined with Fatostatin. A Tumor volume was measured every 3 days. After euthanasia, images of the tumors (B), body weight (C), and tumor weight (D) were evaluated. *p < 0.05, **p < 0.01, and ***p < 0.001: vector vs. HN1 OE. ## p < 0.01 and ###p < 0.001: HN1 OE vs. HN1 OE plus Fatostatin. “Vector” refers to the control vector plasmid, “HN1 OE” to HN1 overexpression, and “Fatostatin” to the SREBP inhibitor.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Inhibition, Transfection, Control, Over Expression, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 12 Schematic representation of HN1-mediated inhibition of tumorigenesis of HCC. HN1 suppression deactivates the Akt pathway, leading to reduced levels of mature SREBP in the nucleus, thereby inhibiting lipogenesis and ultimately suppressing hepato- cellular carcinoma (HCC) tumorigenesis.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Inhibition

Journal: MedComm

Article Title: Profiling of SARS‐CoV‐2 neutralizing antibody‐associated antigenic peptides signature using proteome microarray

doi: 10.1002/mco2.361

Figure Lengend Snippet: Immunoglobulin G (IgG) antibodies against SARS‐CoV‐2 in recovered patients with COVID‐19 were analyzed using a proteome microarray. (A) Schematic diagram of the proteome microarray used for the detection of SARS‐CoV‐2 peptide‐IgGs. (B) Spearman's correlation for the ELISA and proteome microarray. The X ‐axis indicates the rank of N‐IgG fluorescence intensity for each sample detected using proteome microarray. The Y ‐axis indicates the rank of N‐IgG absorbance for each sample detected by ELISA. (C) Research design and participants' information. The plasma samples were collected from patients who had recovered from COVID‐19 during three visits. (D) Neutralizing antibody (NAb) titers against SARS‐CoV‐2 in patients with COVID‐19 during the three visits. The box outlines represent the 25th–75th percentiles and the middle lines indicate the median values. The whiskers indicate 1.5 times the interquartile range (values greater than or lower than the extremes were regarded as outliers). F1, Follow‐up 1; F2, Follow‐up 2; N, NAbs‐negative; P, NAbs‐positive; PN, NAbs‐negative conversion during follow‐up; NP, NAbs‐positive conversion during follow‐up.

Article Snippet: SARS‐CoV‐2 proteins, including S (Val16–Pro1213), S1 (Val16‐Arg685), S2 (Ser686‐Pro1213), RBD (Arg319‐Phe541), and N (Met1‐Ala419), were expressed in insect cells or human HEK293 cells (Sino Biological).

Techniques: Microarray, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: MedComm

Article Title: Profiling of SARS‐CoV‐2 neutralizing antibody‐associated antigenic peptides signature using proteome microarray

doi: 10.1002/mco2.361

Figure Lengend Snippet: The potency of S‐82 and ORF10‐3 immunoglobulin G (IgG) for SARS‐CoV‐2 neutralizing antibody (NAb) seropositivity evaluation. (A) Receiver operating characteristic (ROC) curve of logistic regression to evaluate NAbs seropositivity in the testing set using S82‐IgG and ORF10‐3‐IgG. The X ‐axis indicates the false positive rate, and the Y ‐axis indicates the true positive rate. ROC curves (blue) were generated after 100 runs of computational cross‐validation and the mean ROC curve (red) was generated by averaging all of the ROC curves. The mean and 95% confidence interval (CI) of the areas under the curves (AUCs) after 100 runs of computational cross‐validation were also calculated. (B) The mean accuracy of different IgG combinations for SARS‐CoV‐2 NAbs seropositivity evaluation in support vector machine classifiers with 10‐fold cross‐validation. The two IgGs combined in the classifier were referred to Antibody 1 and Antibody 2. (C and D) The level of IgGs against S‐82 (C) and ORF10‐3 (D) at the last visit before F2 across groups. The dots indicate the IgG absorbance in the proteome microarray. The box outlines represent the 25th–75th percentiles and the middle lines indicate the median values. The whiskers indicate 1.5 times the interquartile range (values greater than or lower than the extremes were regarded as outliers). The p values in the Kolmogorov–Smirnov test are shown. OD 532 = fluorescent signal intensity at 532 nm.

Article Snippet: SARS‐CoV‐2 proteins, including S (Val16–Pro1213), S1 (Val16‐Arg685), S2 (Ser686‐Pro1213), RBD (Arg319‐Phe541), and N (Met1‐Ala419), were expressed in insect cells or human HEK293 cells (Sino Biological).

Techniques: Generated, Plasmid Preparation, Microarray

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 2 Validation of clusterin upregulation by semi-quantitative reverse transcription–PCR, western blot analysis and immunofluorescence microscopy. (a) Semi-quantitative reverse transcription–PCR analysis (bottom panels) of the clusterin transcript (CLU) in BRI-JM01 cells induced after 2, 4, 6 and 24 h of TGF-b1 (100 pM) exposure confirming the clusterin transcript modulation observed in the microarray experiments (The graph underneath represents normalized data obtained from the microarray experiments. Bars represent the t-test P-value of 4–6 experiments performed for each time point.) The Eef1a-1 transcript (top panel), which does not change upon TGF-b1 treatment, was used as loading control. (b) Western blot analysis of whole-cell lysates (WCL) from BRI-JM01 cells grown in the absence or presence of 100 pM TGF-b1 (24 h) confirming the upregulation of clusterin and showing the uncleaved precursor form (pCLU) of clusterin in the WCL, and the the mature processed form (sCLU) in both the WCL and the CM. Membranes containing the WCL samples were reprobed for b-actin to confirm equal loading. (c) Immunofluorescence microscopy (magnification 1000) of BRI-JM01 cells treated with 100 pM TGF-b1 (24 h) show clusterin to be close to the cell’s outer membrane (top panels) and (d) colocalized with the Golgi marker b-COP (bottom) (red, clusterin; green, b-COP; blue, diamidino phenylindole (DAPI)-stained nuclei; magnification 400). (e) Western blot of the CM confirming the increased levels of secreted clusterin (sCLU) in the medium of BRI-JM01 cells treated with 100 pM TGF-b1 (24 h). CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Biomarker Discovery, Reverse Transcription, Western Blot, Microscopy, Microarray, Control, Membrane, Marker, Staining

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 3 Antibodies against sCLU block the TGF-b1-induced loss of junctional ZO-1 and E-cadherin in BRI-JM01 cells. Immunofluorescence microscopy of ZO-1 in BRI-JM01 cells grown (a) in the absence or presence of 100 pM TGF-b1 with or without antibodies against TGF-b (anti-TGF-b, 10 nM) or clusterin (anti-clu, 8 mg/ml), or (b) in CM from untreated BRI-JM01 cells (CM-CTL) or from cells treated with 100 pM TGF-b1 for 24 h (CM-TGF-b1) both in the absence or presence of antibodies against (anti-TGF-b, 10 nM) or clusterin (anti-clu, 8 mg/ml). For all the panels: red, ZO-1; blue, diamidino phenylindole (DAPI)-stained nuclei (magnification: 400). (c) Flow cytometric evaluation of the cell-surface levels of E-cadherin expressed by BRI-JM01 cells exposed to TGF-b1 (100 pM) in the absence or presence of clusterin polyclonal IgG (anti-clu, 8 mg/ml). Cell populations expressing high ( þ ) and low () levels of cell-surface E-cadherin are indicated. CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b; ZO, zona occludens.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Blocking Assay, Microscopy, Staining, Expressing

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 4 Purified clusterin promotes a spindle-shaped morphol- ogy and loss of junctional ZO-1 in BRI-JM01 cells. (a) Coomassie blue-stained gel under non-reducing (DTT (dithiothreitol)) and reducing ( þ DTT) conditions of purified recombinant human clusterin (b) expressed and secreted by HEK-293-E6 cells. (b) BRI- JM01 cells treated with 200 nM purified clusterin (24 h) show a spindle-shaped morphology (top; magnification 40) and loss of tight-junctional ZO-1 (red, bottom; magnification 400). HEK, human embryonic kidney; ZO, zona occludens.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Staining, Recombinant

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 6 Clusterin polyclonal IgG inhibits the invasive behavior of cell lines other than the BRI-JM01 cell line. Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits (a) the stellate morphology of 4T1 and PC3 tumor cells when cultured in Matrigel for 3 weeks (magnification 40). (b) Western blot analysis confirming the presence of sCLU in the CM of 4T1, NMuMG and PC3 cells. (c) Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits the Matrigel invasion of 4T1, NMuMG and PC3 cell lines ing a Transwell invasion assay. Results (expressed relative to non-treated cells) are shown as the average (±s.d.) of two independent experiments. (d) Clusterin polyclonal IgG (anti-clu, 8 mg/ml) does not significantly affect the growth-inhibitory response induced by TGF-b1 in 4T1, NMuMG and PC3 cells. Results are expressed relative to non-treated cells and are shown as the average (±s.d.) of two independent experiments performed in triplicate. (e) Western blot analysis showing the presence of sCLU in the CM of MDA-MB231LM2 cells (left panel). Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits the Matrigel invasion of these cells in a Transwell assay (right panel). Results (expressed relative to non-treated cells) are shown as the average (±s.d.) of two independent experiments. CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Cell Culture, Western Blot, Transwell Invasion Assay, Transwell Assay

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 7 Clusterin monoclonal antibodies inhibit the motility of 4T1 cells and significantly reduce 4T1 cell metastasis to lungs after orthotopic implantation. (a) Evaluation of the ability of in-house-generated clusterin monoclonal antibodies 11E2, 16B5, 16C11 and 20G3 at a concentration of 8 mg/ml (black bars), and a commercially available clusterin monoclonal antibody B5 and anti-peptide polyclonal IgG, C18 (white bars), to block 4T1 cell motility in a BCSM assay. The average motility (±s.d.) was determined by measuring ink clearance in 10 independent microscopic fields (per treatment) and is expressed as average ink clearance/cell/24 h relative to non-treated control cells (gray bar). The hatched line depicts the cut-off that we used to define an antibody as having neutralizing activity, which was based on the degree of inhibition caused by B5 antibody. (b) Experimental design. 4T1 cells (4 104) were injected in the left # 4 inguinal mammary gland. Animals were treated (5 mg/kg) with neutralizing (16B5, 16C11 and 11E2) clusterin antibody, non-neutralizing (20G3) clusterin antibody or saline (control) thrice a week (intraperitoneally), starting the day of cell implantation. Mice were killed on day 28. (c) The number of macroscopically grossly visible lung metastasis in the mice were quantified 28 days post tumor cell implantation and statistically analysed using the non-parametric Mann–Whitney U-test (n ¼ 10 for the saline, 20G3 and 11E2 groups; n ¼ 9 for the 16B5 and 16C11 groups). BCSM, black cellular spreading and motility.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Bioprocessing, Generated, Concentration Assay, Blocking Assay, Control, Activity Assay, Inhibition, Injection, Saline, MANN-WHITNEY

Journal: Oncotarget

Article Title: Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

doi: 10.18632/oncotarget.8290

Figure Lengend Snippet: ONCOMINE™ database analysis of DPYSL3 gene expression

Article Snippet: Antibodies for CRMP4 (sc-100323), Actin (sc-1616), Ki-67 (sc-23900) and DPYSL3 siRNA (sc-44487) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques:

Journal: Oncotarget

Article Title: Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

doi: 10.18632/oncotarget.8290

Figure Lengend Snippet: A. Public dataset was extracted from Oncomine™ and graphed in to three groups. The numbers in white indicate the median value and patient case number is indicated on x-axel. B. cDNA microarray dataset from a previously published report was re-analyzed. Data represent the Mean from different patient groups, including benign prostate specimens (normal, n = 5), primary cancers (n = 23), tumors after hormone therapy (n = 17), metastasis (n = 9) and castration-resistant tumors (n = 3). The errors bars indicate the standard error of mean (SEM). The asterisk indicates a significant difference compared to other groups (p < 0.05, student's t -test). C. Quantitative analysis of DPYSL3 variants in prostate cancers were conducted using total RNAs extracted from frozen tumor specimens and the individually matched nonmalignant compartments, as described . The expression levels of DPYSL3 variants were normalized against the epithelium-specific gene KRT18 before the relative values were calculated. The relative ratio of gene expression level in malignant compared to benign tissues was presented as fold induction. Error bar represents the SEM. The asterisk indicates a significant difference compared to the ‘normal’ group (p < 0.05, student's t -test). D. Western blot assays were used to evaluate CRMP4 protein expression in 3 pairs of prostate specimens. Cell lysates from 293T cells overexpressing CRMP4a and CRMP4b were used to as positive control. Actin blot served as protein loading control. Data represent two separate experiments.

Article Snippet: Antibodies for CRMP4 (sc-100323), Actin (sc-1616), Ki-67 (sc-23900) and DPYSL3 siRNA (sc-44487) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques: Microarray, Expressing, Gene Expression, Western Blot, Positive Control, Control

Journal: Oncotarget

Article Title: Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

doi: 10.18632/oncotarget.8290

Figure Lengend Snippet: DPYSL3 saRNA target sequence and genomic location on Chromosome 5

Article Snippet: Antibodies for CRMP4 (sc-100323), Actin (sc-1616), Ki-67 (sc-23900) and DPYSL3 siRNA (sc-44487) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques: Sequencing

Journal: Oncotarget

Article Title: Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

doi: 10.18632/oncotarget.8290

Figure Lengend Snippet: DPYSL3 saRNA suppresses tumor metastasis in vivo

Article Snippet: Antibodies for CRMP4 (sc-100323), Actin (sc-1616), Ki-67 (sc-23900) and DPYSL3 siRNA (sc-44487) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques:

Journal: Oncotarget

Article Title: Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

doi: 10.18632/oncotarget.8290

Figure Lengend Snippet: A. Representing H&E section images from major organs as indicated harvested from animals received the APT-saV2-9 or the scramble control. The green arrows indicate metastatic cancer cells in left lung (a), liver (b) and lymph node (c) sections from animals received the control saRNA. No metastatic cancer cells were seen in kidney sections or other organs. B. The whole right lobes of lung harvested from animals treated with the scramble or sa V2-9 conjugates were homogenized and protein extracts were collected for luciferase activity measurement. The readings were normalized with the corresponding protein concentrations expressed as relative LUC reading/mg lung proteins. Data presented are MEAN ± SEM and the asterisk indicates a significant difference compared to the scramble control (P < 0.05, Student's t-test). C. Total RNAs were extracted from xenograft tissues and subjected to qPCR analysis for DPYSl3 gene expression. After normalized with KRT18 gene expression levels, relative fold induction against the value in the samples from the scramble control group was calculated and presented as MEAN ± SEM. The asterisk indicates a significant difference compared to the scramble control (p < 0.05, student's t -test). D. Total proteins were extracted from xenograft tumors and subjected to western blot assays for CRMP4a protein expression. Actin blot served as protein loading control. E. Xenograft tumors obtained from animals treated with the scramble or saV2-9 conjugates were processed for H&E staining, Ki-67 and BrdU immunohistochemical staining. Representing microscopic images were taken from a 200x magnification.

Article Snippet: Antibodies for CRMP4 (sc-100323), Actin (sc-1616), Ki-67 (sc-23900) and DPYSL3 siRNA (sc-44487) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques: Control, Luciferase, Activity Assay, Gene Expression, Western Blot, Expressing, Staining, Immunohistochemical staining

Journal: Science Advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: ( A ) KSR1 C809Y incorporates phosphorylated ERK. HEK293T cells were transfected with Flag-tagged wild-type (wt) or C809Y KSR1 (1.5 μg) and stimulated with EGF (50 ng/ml, 5 min) where indicated (+) after 18 hours of starvation (−). KSR1-associated proteins were determined by coimmunoprecipitation upon anti-Flag immunoprecipitation (IP: Flag) and subsequent Western blotting. TL, total lysate. ( B ) C809Y binds to phosphorylated ERK in live cells. Ectopic Flag-tagged C809Y interaction with endogenous ERK, determined by PLA in HeLa cells after starvation, in starved (st) or EGF-treated cells. Scale bar, 10 μm. ( C ) C809Y can homodimerize. HEK293T cells were transfected with the indicated Glu- and Flag-tagged KSR1 constructs and EGF-stimulated where indicated (+). Immunoprecipitations performed with a specific antibody (IP) or with preimmune serum (PI). un, untransfected cells. ( D ) KSR1 double mutant fails to bind phosphorylated ERK. Coimmunoprecipitation assay in HEK293T cells transfected with the indicated Flag-tagged KSR1 constructs, in starved cells (−) or upon EGF stimulation where indicated (+). ( E ) KSR1 double mutant fails to bind phosphorylated ERK in vivo. HeLa cells were transfected with the indicated KSR1 constructs (2 μg). PLA as in (B), in EGF-stimulated cells. Scale bar, 10 μm. (A and D) Figures show signal intensity relative to the levels found in untreated cells. All the results shown are representative of three to five independent experiments.

Article Snippet: shRNAs against human KSR1 (TRCN 006226, TRCN 006227, TRCN 006229, and TRCN 006230 XM 290793), human KSR2 (TRCN 007062, TRCN 335901, TRCN 199619, TRCN 199136, and TRCN 195374 NM 173593), and human IQGAP1 (TRCN 47485, TRCN 47487, TRCN 298928, TRCN 298930, and TRCN 298931) were obtained from Sigma-Aldrich. siRNA against KSR1 (no. sc-35762) and siRNA against IQGAP1 (no. sc-35700) were purchased from Santa Cruz Biotechnology. siRNAs and shRNAs were transfected with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, no. 13778150) following the manufacturer’s directions. pCDNA3 MYC IQGAP1 WW, ∆IQ, ∆CHD, N1, N2, N, and C were generated by D. Sacks; pEF-BOS MYC IQGAP1 was supplied by K. Kaibuchi; pCDNA3 Ksr1 GLU was provided by W. J. Fantl; pHis parallel KSR1, pCMV FLAG KSR1 wild type, C809Y, ASAP, 176, 305, 402, 521, and ∆N were a gift from J. Lozano. pCEFL HA HRAS V12 has been previously described ( ).

Techniques: Transfection, Immunoprecipitation, Western Blot, Construct, Mutagenesis, Co-Immunoprecipitation Assay, In Vivo

Journal: Science Advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: ( A ) KSR1 C809Y is active in KSR1 −/− MEFs. Left: cPLA 2 activity in KSR1 −/− MEFs transfected with the indicated KSR1 constructs (2 μg) under starvation conditions and after EGF stimulation (50 ng/ml, 5 min). Data show means ± SEM for two independent experiments, relative to the value of KSR1 wt. P values: ** P < 0.01 by two-tailed unpaired Student’s t test. Right: KSR1 construct expression levels. ( B ) IQGAP1 expression in wt and KSR −/− MEFs. ( C ) IQGAP1 and KSR1 interaction in vivo. Endogenous IQGAP1 or KSR1 was immunoprecipitated from HEK293T, starved for 18 hours (−), or EGF-stimulated, and the coimmunoprecipitating scaffold was detected by WB. PI, immunoprecipitation using preimmune serum. ( D ) IQGAP1 and KSR1 interaction in vitro. Pull-down using glutathione S -transferase (GST)–IQGAP1 with a bacterially expressed, purified, full-length KSR1. ( E ) IQGAP1 and KSR1 interaction in live cells. Association of ectopic Flag-KSR1 and Myc-IQGAP1 (1 μg each), determined by PLA in HeLa cells after starvation, in starved (st) or EGF-treated cells. Control, untransfected cells. Scale bar, 10 μm (see also fig. S1). ( F ) Association of endogenous KSR1 and IQGAP1 in the indicated proliferating tumor cell lines, as determined by coimmunoprecipitation upon anti-IQGAP1 immunoprecipitation (IP). All the results shown are representative of three to five independent experiments.

Article Snippet: shRNAs against human KSR1 (TRCN 006226, TRCN 006227, TRCN 006229, and TRCN 006230 XM 290793), human KSR2 (TRCN 007062, TRCN 335901, TRCN 199619, TRCN 199136, and TRCN 195374 NM 173593), and human IQGAP1 (TRCN 47485, TRCN 47487, TRCN 298928, TRCN 298930, and TRCN 298931) were obtained from Sigma-Aldrich. siRNA against KSR1 (no. sc-35762) and siRNA against IQGAP1 (no. sc-35700) were purchased from Santa Cruz Biotechnology. siRNAs and shRNAs were transfected with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, no. 13778150) following the manufacturer’s directions. pCDNA3 MYC IQGAP1 WW, ∆IQ, ∆CHD, N1, N2, N, and C were generated by D. Sacks; pEF-BOS MYC IQGAP1 was supplied by K. Kaibuchi; pCDNA3 Ksr1 GLU was provided by W. J. Fantl; pHis parallel KSR1, pCMV FLAG KSR1 wild type, C809Y, ASAP, 176, 305, 402, 521, and ∆N were a gift from J. Lozano. pCEFL HA HRAS V12 has been previously described ( ).

Techniques: Activity Assay, Transfection, Construct, Two Tailed Test, Expressing, In Vivo, Immunoprecipitation, In Vitro, Purification

Journal: Science Advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: ( A ) The KSR1-binding domain is located in the C-terminal region of IQGAP1. HEK293T cells were transfected with the indicated Myc-tagged IQGAP1 constructs and Flag-tagged KSR1 (1.5 μg) and stimulated with EGF (50 ng/ml, 5 min). After 18-hour starvation, cells were stimulated with EGF (50 ng/ml, 5 min). The IQGAP1 constructs associated with KSR1 were determined by anti-Flag immunoprecipitation with subsequent anti-Myc immunoblotting. PI, immunoprecipitation using preimmune serum. ( B ) The IQGAP1-binding domain is located between amino acids 402 and 521 of KSR1. As in (A), cells were transfected with the indicated Flag-tagged KSR1 constructs and Myc-tagged IQGAP1. ( C ) ERK and IQGAP1 bind to the same region of KSR1. Lysates from HEK293T cells starved (−) or EGF-stimulated (+) were pulled down using GST-KSR1 301-600. Associated proteins were revealed by immunoblotting. ( D ) Effects of IQGAP1 down-regulation on KSR1-bound phosphorylated ERK, as determined by coimmunoprecipitation upon anti-KSR1 immunoprecipitation from wt and IQGAP1 CRISPR-Cas9–knockout (KO) SKMEL2 cells. Figures show signal intensity relative to the levels found in wt cells. All the results shown are representative of three to five independent experiments (see also fig. S2).

Article Snippet: shRNAs against human KSR1 (TRCN 006226, TRCN 006227, TRCN 006229, and TRCN 006230 XM 290793), human KSR2 (TRCN 007062, TRCN 335901, TRCN 199619, TRCN 199136, and TRCN 195374 NM 173593), and human IQGAP1 (TRCN 47485, TRCN 47487, TRCN 298928, TRCN 298930, and TRCN 298931) were obtained from Sigma-Aldrich. siRNA against KSR1 (no. sc-35762) and siRNA against IQGAP1 (no. sc-35700) were purchased from Santa Cruz Biotechnology. siRNAs and shRNAs were transfected with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, no. 13778150) following the manufacturer’s directions. pCDNA3 MYC IQGAP1 WW, ∆IQ, ∆CHD, N1, N2, N, and C were generated by D. Sacks; pEF-BOS MYC IQGAP1 was supplied by K. Kaibuchi; pCDNA3 Ksr1 GLU was provided by W. J. Fantl; pHis parallel KSR1, pCMV FLAG KSR1 wild type, C809Y, ASAP, 176, 305, 402, 521, and ∆N were a gift from J. Lozano. pCEFL HA HRAS V12 has been previously described ( ).

Techniques: Binding Assay, Transfection, Construct, Immunoprecipitation, Western Blot, CRISPR, Knock-Out

Journal: Science Advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: ( A ) IQGAP1 interacts with KSR1 C809Y. Coimmunoprecipitation assay from HEK293T cells cotransfected with the indicated Flag-tagged KSR1 constructs (1.5 μg each), upon EGF stimulation (50 ng/ml, 5 min) where indicated (+), after 18-hour starvation (−). ( B ) Effects of IQGAP1 depletion on phosphorylated ERK binding to KSR1 C809Y. Coimmunoprecipitation assay in KSR1 −/− MEFs expressing the indicated Flag-tagged KSR1 constructs and transfected with shRNA against IQGAP1 where indicated (+) (1 μg), following EGF stimulation (+). ( C ) IQGAP1 overexpression facilitates KSR1 incorporation of phosphorylated ERK. Coimmunoprecipitation from HEK293T cells transfected with the indicated Flag-tagged KSR1 constructs plus Myc-tagged IQGAP1, in starved cells (−) or upon EGF stimulation where indicated (+). Figures show signal intensity relative to the levels in starved cells. ( D ) KSR1 incorporation of phosphorylated ERK mediated by IQGAP1 KSR-binding and MEK-binding mutants. Coimmunoprecipitation from HEK293T cells transfected with both Flag-tagged KSR1 615/809 and the indicated Myc-tagged IQGAP1 mutants (1 μg each), in starved cells (−) or upon EGF stimulation as shown (+); ev, empty vector. In all cases, immunoprecipitations were performed with a specific antibody (IP) or with preimmune serum (PI). All the results shown are representative of three to five independent experiments (see also fig. S3).

Article Snippet: shRNAs against human KSR1 (TRCN 006226, TRCN 006227, TRCN 006229, and TRCN 006230 XM 290793), human KSR2 (TRCN 007062, TRCN 335901, TRCN 199619, TRCN 199136, and TRCN 195374 NM 173593), and human IQGAP1 (TRCN 47485, TRCN 47487, TRCN 298928, TRCN 298930, and TRCN 298931) were obtained from Sigma-Aldrich. siRNA against KSR1 (no. sc-35762) and siRNA against IQGAP1 (no. sc-35700) were purchased from Santa Cruz Biotechnology. siRNAs and shRNAs were transfected with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, no. 13778150) following the manufacturer’s directions. pCDNA3 MYC IQGAP1 WW, ∆IQ, ∆CHD, N1, N2, N, and C were generated by D. Sacks; pEF-BOS MYC IQGAP1 was supplied by K. Kaibuchi; pCDNA3 Ksr1 GLU was provided by W. J. Fantl; pHis parallel KSR1, pCMV FLAG KSR1 wild type, C809Y, ASAP, 176, 305, 402, 521, and ∆N were a gift from J. Lozano. pCEFL HA HRAS V12 has been previously described ( ).

Techniques: Co-Immunoprecipitation Assay, Construct, Binding Assay, Expressing, Transfection, shRNA, Over Expression, Plasmid Preparation

Journal: Science Advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: ( A ) The role of trans-phosphorylation on BRAF/KSR1 interaction. Coimmunoprecipitation assay from HEK293T cells cotransfected with the indicated Flag-tagged KSR1 constructs (1.5 μg each), upon EGF stimulation (50 ng/ml, 5 min), or when cotransfected with HRAS V12 (1 μg), after 18-hour starvation (−). ( B ) Impact of trans-phosphorylation on KSR1-mediated ERK dimerization. Performed as in (A), ERK dimerization was determined by native electrophoresis. Bands corresponding to ERK monomers and dimers are indicated. ( C ) ERK dimerization in response to KSR1 inactivation and/or IQGAP1 depletion. Determined in endogenous KSR1 immunoprecipitates from cells transfected with HRAS V12 (+) in the presence (+) or absence (−) of an shRNA against IQGAP1 after 18-hour starvation. Where indicated, cells were treated with APS-2-79 (5 μM, 2 hours). ( D ) Effects of IQGAP1 down-regulation on KSR1-bound phosphorylated ERK, as determined by coimmunoprecipitation upon anti-KSR1 immunoprecipitation from wt and IQGAP1 shRNA down-regulated (KD) Cal62 and Hth83 cells. Figures show signal intensity relative to the levels in wt cells. ( E ) ERK transphosphorylation in IQGAP1 MEK–binding mutant. HEK293T cells were transfected with the indicated MYC-tagged IQGAP1 constructs and stimulated with EGF where shown (+). ( F ) Effects of KSR1 down-regulation on IQGAP1-bound phosphorylated ERK, as determined by coimmunoprecipitation upon anti-IQGAP1 immunoprecipitation from wt and KSR1 knock-out ( −/− ) MEFs. In all cases, immunoprecipitations were performed with a specific antibody (IP) or with preimmune serum (PI). All the results shown are representative of three independent experiments.

Article Snippet: shRNAs against human KSR1 (TRCN 006226, TRCN 006227, TRCN 006229, and TRCN 006230 XM 290793), human KSR2 (TRCN 007062, TRCN 335901, TRCN 199619, TRCN 199136, and TRCN 195374 NM 173593), and human IQGAP1 (TRCN 47485, TRCN 47487, TRCN 298928, TRCN 298930, and TRCN 298931) were obtained from Sigma-Aldrich. siRNA against KSR1 (no. sc-35762) and siRNA against IQGAP1 (no. sc-35700) were purchased from Santa Cruz Biotechnology. siRNAs and shRNAs were transfected with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, no. 13778150) following the manufacturer’s directions. pCDNA3 MYC IQGAP1 WW, ∆IQ, ∆CHD, N1, N2, N, and C were generated by D. Sacks; pEF-BOS MYC IQGAP1 was supplied by K. Kaibuchi; pCDNA3 Ksr1 GLU was provided by W. J. Fantl; pHis parallel KSR1, pCMV FLAG KSR1 wild type, C809Y, ASAP, 176, 305, 402, 521, and ∆N were a gift from J. Lozano. pCEFL HA HRAS V12 has been previously described ( ).

Techniques: Co-Immunoprecipitation Assay, Construct, Electrophoresis, Transfection, shRNA, Immunoprecipitation, Binding Assay, Mutagenesis, Knock-Out

Journal: Science Advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: ( A ) The role of IQGAP1 in adipogenesis mediated by KSR1. Micrographs of KSR1 wt and KSR1 −/− MEFs transfected with the indicated KSR1 constructs in the presence of an shRNA against IQGAP1 (1 μg each) where indicated, after 8 days of treatment with MDI. Adipogenesis was revealed by oil-red staining. ( B ) Quantification of the above results. Data show means ± SEM of three independent experiments. P values: * P < 0.05; ns, not significant, by Student’s t test. ( C ) The role of IQGAP1 in RAS-induced senescence mediated by KSR1. KSR1 −/− MEFs were transfected with HRAS V12 and the indicated KSR1 constructs in the presence (+) or absence (−) of an shRNA against IQGAP1. Senescence was scored by β-galactosidase staining. Data show means ± SEM of three independent experiments, relative to the value in cells not transfected with RAS (control). P values: ** P < 0.01; ns, not significant, by Student’s t test. ( D ) Micrographs of KSR1 −/− MEFs transfected with oncogenic RAS and the indicated KSR1 constructs in the presence of shRNA against IQGAP1 where shown (1 μg each). Senescence was revealed by β-galactosidase staining. All the results shown are representative of three to five independent experiments.

Article Snippet: shRNAs against human KSR1 (TRCN 006226, TRCN 006227, TRCN 006229, and TRCN 006230 XM 290793), human KSR2 (TRCN 007062, TRCN 335901, TRCN 199619, TRCN 199136, and TRCN 195374 NM 173593), and human IQGAP1 (TRCN 47485, TRCN 47487, TRCN 298928, TRCN 298930, and TRCN 298931) were obtained from Sigma-Aldrich. siRNA against KSR1 (no. sc-35762) and siRNA against IQGAP1 (no. sc-35700) were purchased from Santa Cruz Biotechnology. siRNAs and shRNAs were transfected with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, no. 13778150) following the manufacturer’s directions. pCDNA3 MYC IQGAP1 WW, ∆IQ, ∆CHD, N1, N2, N, and C were generated by D. Sacks; pEF-BOS MYC IQGAP1 was supplied by K. Kaibuchi; pCDNA3 Ksr1 GLU was provided by W. J. Fantl; pHis parallel KSR1, pCMV FLAG KSR1 wild type, C809Y, ASAP, 176, 305, 402, 521, and ∆N were a gift from J. Lozano. pCEFL HA HRAS V12 has been previously described ( ).

Techniques: Transfection, Construct, shRNA, Staining

Journal: Science Advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: ( A to D ) Relative expression levels of KSR1 (A and B) and IQGAP1 (C and D) transcripts in healthy and tumor samples obtained from the microarray datasets GSE15471 (A and C) and GSE16515 (B and D). ( E and F ) Regression graph showing the negative correlation between KSR1 and IQGAP1 transcripts levels in microarray datasets GSE15471 (E) and GSE16515 (F). Dots, violin plots, and regression line colors correspond to tumor (red) and healthy (green) samples from the indicated dataset. *** P < 0.001 (A to D). In (D) and (F), the regression coefficient and P value are indicated in each case (see also fig. S3).

Article Snippet: shRNAs against human KSR1 (TRCN 006226, TRCN 006227, TRCN 006229, and TRCN 006230 XM 290793), human KSR2 (TRCN 007062, TRCN 335901, TRCN 199619, TRCN 199136, and TRCN 195374 NM 173593), and human IQGAP1 (TRCN 47485, TRCN 47487, TRCN 298928, TRCN 298930, and TRCN 298931) were obtained from Sigma-Aldrich. siRNA against KSR1 (no. sc-35762) and siRNA against IQGAP1 (no. sc-35700) were purchased from Santa Cruz Biotechnology. siRNAs and shRNAs were transfected with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, no. 13778150) following the manufacturer’s directions. pCDNA3 MYC IQGAP1 WW, ∆IQ, ∆CHD, N1, N2, N, and C were generated by D. Sacks; pEF-BOS MYC IQGAP1 was supplied by K. Kaibuchi; pCDNA3 Ksr1 GLU was provided by W. J. Fantl; pHis parallel KSR1, pCMV FLAG KSR1 wild type, C809Y, ASAP, 176, 305, 402, 521, and ∆N were a gift from J. Lozano. pCEFL HA HRAS V12 has been previously described ( ).

Techniques: Expressing, Microarray

Journal: Science Advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: ( A ) APS-2-79 does not phenocopy the effect of KSR1/2 ablation. A375 and SKMEL2 cells were transfected with the indicated shRNAs or an empty vector as a negative control (c) or treated with APS-2-79 (5 μM, 48 hours). Staurosporine (0.5 μM, 48 hours) was used as a positive control. Apoptosis was evaluated 48 hours after transfection by scoring annexin V levels. Data show means ± SEM from three independent experiments. P values: **** P < 0.001, *** P < 0.005, ** P < 0.01, and * P < 0.05 by Student’s t test. ( B ) APS-2-79 does not affect phosphorylated ERK levels incorporated to KSR1. Endogenous levels bound to KSR1 C809Y were determined by coimmunoprecipitation analyses in APS-2-79–treated cells, stimulated with EGF (50 ng/ml, 5 min) where shown (+) after 18-hour starvation. ( C ) APS-2-79 does not affect KSR1-IQGAP1 interaction. Association of the endogenous proteins was determined by coimmunoprecipitation analyses. ( D ) IC 50 and IQGAP1/KSR1 ratio in the indicated NRAS-mutant cell lines and correlation between both parameters. Data show mean of three independent experiments. ( E ) Impact of IQGAP1 depletion on the APS-2-79 inhibitory effect on KSR1. As in (C), IQGAP1 was depleted by shRNA where indicated. ( F ) IQGAP1 depletion effects on APS-2-79 efficacy. The indicated tumor cells: wt, IQGAP1 down-regulated using shRNAs (sh), and IQGAP1 down-regulated transfected with an ectopic IQGAP1 (1 μg; sh + IQ) were treated with APS 2-79 where indicated. Data show means ± SEM, % of nonviable cells for three independent experiments. P values: *** P < 0.005, ** P < 0.01, and * P < 0.05 by Student’s t test. Bottom: IQGAP1 levels in the indicated cell lines. In all cases, immunoprecipitations were performed with a specific antibody (IP) or with preimmune serum (PI). All the results shown are representative of three to five independent experiments (see also fig. S4).

Article Snippet: shRNAs against human KSR1 (TRCN 006226, TRCN 006227, TRCN 006229, and TRCN 006230 XM 290793), human KSR2 (TRCN 007062, TRCN 335901, TRCN 199619, TRCN 199136, and TRCN 195374 NM 173593), and human IQGAP1 (TRCN 47485, TRCN 47487, TRCN 298928, TRCN 298930, and TRCN 298931) were obtained from Sigma-Aldrich. siRNA against KSR1 (no. sc-35762) and siRNA against IQGAP1 (no. sc-35700) were purchased from Santa Cruz Biotechnology. siRNAs and shRNAs were transfected with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, no. 13778150) following the manufacturer’s directions. pCDNA3 MYC IQGAP1 WW, ∆IQ, ∆CHD, N1, N2, N, and C were generated by D. Sacks; pEF-BOS MYC IQGAP1 was supplied by K. Kaibuchi; pCDNA3 Ksr1 GLU was provided by W. J. Fantl; pHis parallel KSR1, pCMV FLAG KSR1 wild type, C809Y, ASAP, 176, 305, 402, 521, and ∆N were a gift from J. Lozano. pCEFL HA HRAS V12 has been previously described ( ).

Techniques: Transfection, Plasmid Preparation, Negative Control, Positive Control, Mutagenesis, shRNA

Journal: Science Advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: As demonstrated in KSR1/IQGAP1 complexes in response to HRAS V12 signals or EGF stimulation. Trans-phosphorylation via IQGAP1 can bypass the inhibitory effect of APS-2-79 on KSR1-bound ERK activation.

Article Snippet: shRNAs against human KSR1 (TRCN 006226, TRCN 006227, TRCN 006229, and TRCN 006230 XM 290793), human KSR2 (TRCN 007062, TRCN 335901, TRCN 199619, TRCN 199136, and TRCN 195374 NM 173593), and human IQGAP1 (TRCN 47485, TRCN 47487, TRCN 298928, TRCN 298930, and TRCN 298931) were obtained from Sigma-Aldrich. siRNA against KSR1 (no. sc-35762) and siRNA against IQGAP1 (no. sc-35700) were purchased from Santa Cruz Biotechnology. siRNAs and shRNAs were transfected with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, no. 13778150) following the manufacturer’s directions. pCDNA3 MYC IQGAP1 WW, ∆IQ, ∆CHD, N1, N2, N, and C were generated by D. Sacks; pEF-BOS MYC IQGAP1 was supplied by K. Kaibuchi; pCDNA3 Ksr1 GLU was provided by W. J. Fantl; pHis parallel KSR1, pCMV FLAG KSR1 wild type, C809Y, ASAP, 176, 305, 402, 521, and ∆N were a gift from J. Lozano. pCEFL HA HRAS V12 has been previously described ( ).

Techniques: Activation Assay