Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: PIK3CA mutations contribute to acquired cetuximab resistance in metastatic colorectal cancer patients

doi: 10.1158/1078-0432.CCR-16-2738

Figure Lengend Snippet: Mutations related to acquired cetuximab resistance.

Article Snippet: In Vitro Functional Assays The PIK3CA point mutations identified in ctDNA and FFPE tumor tissue were introduced into the full-length PIK3CA coding sequence using site-directed mutagenesis and were inserted into an expression vector (RC213112, Origene).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: PIK3CA mutations contribute to acquired cetuximab resistance in metastatic colorectal cancer patients

doi: 10.1158/1078-0432.CCR-16-2738

Figure Lengend Snippet: Pre-existing mutations in tumor tissue and presence in plasma before treatment

Article Snippet: In Vitro Functional Assays The PIK3CA point mutations identified in ctDNA and FFPE tumor tissue were introduced into the full-length PIK3CA coding sequence using site-directed mutagenesis and were inserted into an expression vector (RC213112, Origene).

Techniques:

Journal: PLoS Pathogens

Article Title: SAMHD1 phosphorylation and cytoplasmic relocalization after human cytomegalovirus infection limits its antiviral activity

doi: 10.1371/journal.ppat.1008855

Figure Lengend Snippet: a) SAMHD1 levels were analyzed by immunoblotting in cell lysates of HFFs not infected (n.i.) or infected with AD169 at an MOI of 1 for 3 days. Expression of IE1/IE2 viral antigens was used as control for infection, while the p85 subunit of PI3K was used as loading control. A representative experiment out of six is shown. b) The relative amount of SAMHD1 protein, normalized to that of p85, was determined by densitometric analysis and is relative to that of n.i. cells, which was arbitrarily set as 1. Data are expressed as mean ± SE of eight independent experiments. c) SAMHD1 expression in HFFs not infected (n.i.) or infected with HCMV clinical isolates collected from infants affected by congenital HCMV infection (P1, P6, P7 and P10), or with Merlin. Data derive from one representative experiment out of two. d) SAMHD1 expression in HFFs, ARPE-19 and HMVEC cells infected with the indicated HCMV strains. Data derive from one representative experiment out of two, performed at the same time with all conditions. Numbers indicate the relative amount of SAMHD1 protein, determined as in panel b). e) The relative amount of SAMHD1 protein from HFFs infected with the indicated clinical strains was determined as in panel b). Data are expressed as mean ± SE of eight independent experiments. All data derive from cells infected at an MOI of 1 and harvested at 3 dpi. f) PMA-differentiated THP-1 cells not infected or infected with TR at an MOI of 1 for the indicated times. Expression of SAMHD1, viral antigens and p85 was evaluated as above. A representative experiment out of two is shown. g) SAMHD1 expression in HFFs induced to reach confluence (Con), serum-starved (Starv), proliferating (Prol), or infected with AD169 at an MOI of 1 for 3 days. Data derive from one representative experiment out of two. h) Antibody validation of the anti-SAMHD1 antibody by immunocapture MS. Proteins enriched by the antibody towards SAMHD1 ( y -axis) are plotted versus non-specific enrichment by one Rabbit IgG pool ( x- axis). Enriched proteins identified are quantified by label-free quantification and plotted as intensities (log 10 ). **, p < 0.01; ***, p < 0.001; ns, not significant.

Article Snippet: The following antibodies were used in immunoblot: anti-SAMHD1 rabbit polyclonal antibody (Ab) (Proteintech); anti-SAMHD1 rabbit polyclonal Ab specific for the phosphorylated T592 residue (ProSci); rabbit polyclonal anti-p85 subunit of PI3K (N-SH2 domain) and mouse monoclonal antibody (mAb) anti-IE1/IE2 viral proteins (MAB810R; Merck Millipore); mAb anti-UL97 (kindly provided by Thomas Mertens, Ulm University Medical Center, Germany); rabbit anti-Cdk1 (#77055) and mAb anti-tubulin (both from Cell Signaling); rabbit anti-Cdk2 (sc-163) and mAb anti-lamin A (C-3) (both from Santa Cruz).

Techniques: Western Blot, Infection, Expressing, Control, Biomarker Discovery, Quantitative Proteomics

Journal: PLoS Pathogens

Article Title: SAMHD1 phosphorylation and cytoplasmic relocalization after human cytomegalovirus infection limits its antiviral activity

doi: 10.1371/journal.ppat.1008855

Figure Lengend Snippet: a-h) HFFs were transfected with SAMHD1 siRNA or non-targeting siRNA (ctrl). Two days later, cells were either infected with AD169 at an MOI of 1 and harvested at 3 dpi (a-e) or infected at an MOI of 0.1 or 0.05 and harvested at 6 dpi (f-h). a) Levels of SAMHD1 and IE1/IE2 viral protein expression were assayed by immunoblotting. The p85 subunit of PI3K was used as loading control. One representative experiment is shown. b) The relative amount of SAMHD1 protein, normalized to that of p85, was determined by densitometric analysis and is relative to that of n.i./siCtrl cells, which was arbitrarily set as 1. Data are expressed as mean ± SE. c) The percentage of IE+ cells was analyzed by FACS after intracellular staining with a specific anti-IE1/IE2 mAb. FACS plots derive from one representative experiment. d) The percentage of IE+ cells is expressed as mean ± SD, as detected by FACS. e) Cell culture supernatants were assayed for infectious virus production by plaque assays. Results are expressed as mean ± SE. All data in panels a-e derive from five independent experiments. f) Immunoblotting at 6 dpi was performed as described in panel a). One representative experiment out of two is shown. g) The relative amount of SAMHD1 protein was determined as in panel b). Data are expressed as mean ± SE of two independent experiments with cells infected at both MOI. h) Viral titers were measured and expressed as in panel e) and derive from cell culture supernatants of two independent experiments, were HFFs were infected at an MOI of 0.1 or MOI of 0.05 for 6 days. i-j) HFFs were infected with single-cycle VLPs loaded with Vpx (Vpx) or unloaded (ΔVpx) at an MOI of 1, and then the same cells were infected with AD169 at an MOI of 1. At different dpi, cells and supernatants were harvested and subjected to immunoblotting and plaque assays, respectively. i) Immunoblotting of SAMHD1 protein expression. One representative experiment out of three is shown. j) Cell culture supernatants were assayed for infectious virus production by plaque assays. Results are expressed as mean ± SE of four experiments at 2–3 dpi. nt, not treated; n.i., not infected cells; *, p < 0.05; ***, p < 0.001; ns, not significant.

Article Snippet: The following antibodies were used in immunoblot: anti-SAMHD1 rabbit polyclonal antibody (Ab) (Proteintech); anti-SAMHD1 rabbit polyclonal Ab specific for the phosphorylated T592 residue (ProSci); rabbit polyclonal anti-p85 subunit of PI3K (N-SH2 domain) and mouse monoclonal antibody (mAb) anti-IE1/IE2 viral proteins (MAB810R; Merck Millipore); mAb anti-UL97 (kindly provided by Thomas Mertens, Ulm University Medical Center, Germany); rabbit anti-Cdk1 (#77055) and mAb anti-tubulin (both from Cell Signaling); rabbit anti-Cdk2 (sc-163) and mAb anti-lamin A (C-3) (both from Santa Cruz).

Techniques: Transfection, Infection, Expressing, Western Blot, Control, Staining, Cell Culture, Virus

Journal: Nature Communications

Article Title: Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

doi: 10.1038/s41467-021-25341-9

Figure Lengend Snippet: a Percentage of PI3K members ( PTEN , PIK3CA , PIK3CB, and PIK3R1 ) alterations in metastatic castration-resistant prostate cancer cohort (SU2C Cell 2015). b Oncoprint showing PI3K member alterations in all PI3K-altered tumors from the same cohort ( n = 76). c Venn diagram showing the percentage of overlaps among alterations in PIK3CA , PIK3CB , PIK3R1 , and PTEN . d Oncoprint showing PIK3CA , PIK3R1 , and PTEN alterations in a collection of PDOs. e Western blot showing pAKT level and PTEN status in a collection of PDOs. f Western blot showing AR and pAKT levels in PDOs treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. Quantification of pAKT473 and pAKT308 normalized to β-actin. g Western blot showing pAKT levels in isogenic PDO pairs MSK-PCa15 ( PTEN -intact, PTEN -KO) and MSK-PCa16 ( PTEN -intact, PTEN -KO) treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. h Western blot showing pAKT levels in isogenic LNCaP-EV and LNCaP- PTEN pairs treated with doxycycline (500 ng/mL) for 8 h for PTEN induction, followed by either BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. All assays were performed with three biological replicates. Source data are provided as Source data file.

Article Snippet: pLenti-Lentiviral vector (pLenti-C-Myc-DDK-P2A-Neo) (Origene, PS100081) was used to express PIK3CA wild-type (Origene, RC213112), PIK3CA mutant (E545K) (Origene, RC400348), and PIK3CB wild-type (Origene, RC215433).

Techniques: Western Blot

Journal: Nature Communications

Article Title: Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

doi: 10.1038/s41467-021-25341-9

Figure Lengend Snippet: a – c Western blot showing pAKT levels in CWR22Pc, LNCaP cells, or MSK-PCa1 organoids with ectopic expression of DDK-tagged p110α E545K or p110β E1051K mutants. d Western blot showing pAKT levels in LNCaP-EV, LNCaP-p110α E545K , or LNCaP-p110β E1051K cells treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. e CellTiter-Glo assay showing cell viability in LNCaP-EV, LNCaP-p110α E545K , or LNCaP-p110β E1051K cells treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 7 days. f Western blot showing pAKT levels in CWR22Pc-EV, CWR22Pc-p110α E545K , or CWR22Pc-p110β E1051K cells treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. g CellTiter-Glo assay showing cell viability in CWR22Pc-EV, CWR22Pc-p110α E545K , or CWR22Pc-p110β E1051K cells treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 7 days. h Western blot showing levels of pAKT and pS6 in LNCaP-EV (empty vector) or LNCaP-p110α WT (ectopic expression of DDK-tagged p110α WT ) cells cultured in regular 10% FBS-RPMI treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. Source data are provided as Source data file. All assays were performed with three biological replicates. **** p < 0.0001, n.s: not significant, e : one-way ANOVA compared to BYL719 group, g : one-way ANOVA compared to AZD8186 group, error bar represents mean values ± SD.

Article Snippet: pLenti-Lentiviral vector (pLenti-C-Myc-DDK-P2A-Neo) (Origene, PS100081) was used to express PIK3CA wild-type (Origene, RC213112), PIK3CA mutant (E545K) (Origene, RC400348), and PIK3CB wild-type (Origene, RC215433).

Techniques: Western Blot, Expressing, Glo Assay, Plasmid Preparation, Cell Culture

Journal: Nature Communications

Article Title: Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

doi: 10.1038/s41467-021-25341-9

Figure Lengend Snippet: a Western blot showing levels of AKT signaling in MSK-PCa12 organoids treated with ipatasertib (500 nM) or BYL719 (1 µM) + AZD8186 (250 nM) for 4 h. b CellTiter-Glo assay showing cell viability of MSK-PCa12 organoids treated with BYL719 (1 µM) + AZD8186 (250 nM), enzalutamide (10 µM), ipatasertib (500 nM), enzalutamide + ipatasertib, or Veh, Day 7. c Growth of MSK-PCa12 tumors in SCID mice treated with enzalutamide (10 mg/kg), ipatasertib (50 mg/kg), BYL719 (25 mg/kg) + AZD8186 (75 mg/kg), ipatasertib + enzalutamide, BYL719 + AZD8186 + enzalutamide or Veh. Some of mice were castrated when tumor reached ~200 mm 3 ( n = 5 mice per group) *** p -value = 0.0011. d Growth of CWR22Pc tumors in SCID mice treated with BYL719 (25 mg/kg), ipatasertib (50 mg/kg) or Veh. Some of mice were castrated when tumor reaches ~200 mm 3 ( n = 5 mice per group) Castration + BYL719 vs. Castration + Ipatasertinib, week 2: p -value = 0.045, week 3: p -value = 0.0086, week 4: p -value = 0.012, week 5: p -value = 0.046. e Western blot showing PI3K/AKT/MAPK signaling in CWR22Pc tumors from ( d ). f – h Heat map showing drug sensitivity of ipatasertib (500 nM) or BYL719 (1 µM) + AZD8186 (250 nM) on day 7, in cells characterized as PTEN wild type with PIK3CA mutants or RTK hyperactive ( f ), PIK3CA mutants with PTEN -loss ( g ), and PIK3CA wild type with PTEN -loss ( h ). Genetically manipulated lines are isogenic pairs. i Mechanism model of PI3K pathway alterations and response to clinically available therapeutic agents. All assays were performed with three biological replicates. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, n.s: not significant, b : one-way ANOVA compared to Veh group, error bar represents mean values ± SD. c , d : multiple t -test, two-sided error bar represents mean values ± SEM.

Article Snippet: pLenti-Lentiviral vector (pLenti-C-Myc-DDK-P2A-Neo) (Origene, PS100081) was used to express PIK3CA wild-type (Origene, RC213112), PIK3CA mutant (E545K) (Origene, RC400348), and PIK3CB wild-type (Origene, RC215433).

Techniques: Western Blot, Glo Assay

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Chemokine (CXC Motif) Ligand 12/Chemokine (CXC Motif) Receptor 4 Axis (CXCL12/CXCR4)-mediated Cell Migration by Targeting Mammalian Target of Rapamycin (mTOR) Pathway in Human Gastric Carcinoma Cells *

doi: 10.1074/jbc.M111.302299

Figure Lengend Snippet: Compounds targeting PI3K/mTOR pathway inhibited cell motility stimulated by CXCL12. Motility of MKN-45 cells in the presence of rapamycin (10 nm; A), PI-103 (10 nm; B), or PP-242 (100 nm; C) was determined by the classic Transwell assay as described under “Materials and Methods.” Cells migrating to the lower aspect of the Boyden chamber filter were stained and photographed. Top, representative images of at least three independent experiments. Bottom, quantitative analysis of cell migration. Columns indicate mean of three experiments. Bars, S.D. D, down-regulation of Raptor impeded CXCL12-induced MKN-45 cells migration. After transfection with respective siRNA for 48 h, MKN-45 cells were put into a Transwell assay, and the cells that migrated to the lower chamber were detected after 24 h. Upper panel, down-regulation of Raptor or Rictor after transfection with respective siRNAs. Lower panel, quantitative analysis of cell migration. Columns indicate the mean of three experiments; Bars, S.D. Ctrl, control. NC, negative control.

Article Snippet: Transfection of siRNA The synthetic siRNAs targeting p110 catalytic subunit, Raptor, and Rictor were purchased from Shanghai GenePharma Co., Ltd with sequences as follows: 5′-GGUGGACCACGAAGAGUUATT-3′ (p110α); 5′-CGCAUUUGGUGGACCCAAATT-3′ (p110β); 5′- CAGCGGAGAGCUCAUCAACAATT-3′ (Raptor siRNA1; 5′- GACACGGAAGAUGUUCGACAATT-3′ (Raptor siRNA2); 5′- GCGAGCUGAUGUAGAAUUATT-3′ (Rictor siRNA1); and 5′- GGGAAUACAACUCCAAAUATT-3′ (Rictor siRNA2).

Techniques: Transwell Assay, Staining, Migration, Transfection, Control, Negative Control