Journal: Molecular cancer

Article Title: Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling.

doi: 10.1186/s12943-015-0415-0

Figure Lengend Snippet: Fig. 1 Phenotypic screening in primary NSCLC tumour cells. a Images of cells derived from NSCLC primary tumour #1 cultured in three different conditions. b Effects of the scFv-Fc antibody panel upon NSCLC tumour #1 cell growth in three culture conditions, measured by Cell-Titre Glo (CTG) luminescence signal. Each antibody was individually dosed (without normalising concentrations) and cells were grown in 96-well plates. Positive (anti-IGF1R) and negative control antibodies were dosed in multiple replicates on each plate to establish consistency between plates. Each data point indicates a single well. Black horizontal bars indicate the average value for a sample class. c Scatter plot comparing the effects of scFv-Fc antibodies on NSCLC tumour #1 cell growth grown in spheroids and in standard monolayer cultures. Each data point indicates a single antibody (or replicate of the controls). The dashed box indicates a group of antibodies that strongly inhibited growth of cell monolayers but not spheroids. The solid-line box indicates a group of antibodies with a weak inhibitory effect in both spheroids and monolayers. The orange-coloured datapoint represents an antibody that was later shown to bind CDCP1 (αCDCP1-Ab3 in Fig. 2)

Article Snippet: After blocking, the NCI-H358 cells were stained for 30 min on ice with either DARPin-Fcs at 1 μg/106 cells; allophycocyanin conjugated anti-CDCP1 antibody (R&D Systems FAB26662A/Lot LVQ0109021X) at 10 μL/106 cells, or an isotype control antibody at 1 μg/106 cells.

Techniques: Derivative Assay, Cell Culture, Negative Control

Journal: bioRxiv

Article Title: Modeling signaling-dependent pluripotent cell states with boolean logic can predict cell fate transitions

doi: 10.1101/115683

Figure Lengend Snippet: (a) Predicted population-level gene expression levels (left panel) and qRT-PCR-measured relative expressions (right panel; shown in fold-change from the levels of 2iL) of OSN and lineage markers. Data represents the mean and s.e.m. of three or four biological replicates; the differences between 2iJ and 2iJ+B were examined using a 2-tailed unpaired Student’s t test and asterisks indicate *p < 0.1 and ** p <0.05. (b) In silico subpopulation analysis via threshold-based characterization for individual SCCs under the input condition of 2i-L+B-A. Stable grouped profiles enriched as either PSC, TE, ME, PE or Epiblast-like subpopulations were traced in color-coded circles. The circles with solid line indicate SCCs with no outgoing edges (sustainability score =1.0), and those with dashed line indicate SCCs with lower sustainability. (c) Confocal images of immuno-staining of mESCs for Oct4/Cdx2/Gata4 (top) or Oct4/Cdx2/Brachyury (T) (bottom panels) cultured in the given conditions for 2 days. (d) Quantification of frequency of subpopulations that exhibit features of differentiation lineages. Data are means of three replicates and the results were confirmed in two independent studies. (e) qRT-PCR for pluripotency and extended TE-lineage maker genes. TSCs and mESCs after culture in each condition were compared. Data represents the mean of three replicates. (f) Flow cytometry histograms showing fluorescence intensity of CDCP1 and CD40 in individual samples of mESC in LS, TS, and mESCs cultured in 2iJ+B-A for 2 days. Percentage listed is that of positive cells in the 2iJ+B-A condition. (g) PCA plot of RNA-seq data for the top 40% of genes that show highest variance across all samples. Distinct cell types and conditions are indicated with different colors. Circles include day2 and day5 samples for 2i-L and 2iJ conditions, and two day 2 samples, day 5 and 11 for 2iJ+B-A condition. Diamonds indicate stable cell type no culture time defined. Meso indicates mesoderm progenitors. (h) Comparison of predicted gene expression levels and RNAseq-measured gene counts in 2iJ+B-A relative to 2iJ (Equivalent to 2i-L in simulation) for 29 genes involved in the model. The experimental mean relative gene expressions of day2 samples for the two conditions and the mean relative predicted levels are shown. Black dots indicate genes significantly up- or down-regulated (p < 0.05) in three 2iJ+B-A-treated samples compared with two 2iJ-treated samples. Genes in blue are up-regulated in TSC, and those in red are down-regulated in TSC compared with 2iJ-treated samples. (i) Left panel: In vivo lineage contribution frequency and chimera efficiency of H2B-GFP ESCs treated with either 2iL or 2iJ in the presence of serum. Lineage contribution efficiencies were calculated as number of chimeras with cells in Epiblast (EPI) or TE positions/total number of chimeras. Note that cells scored as “TE-position” did not express TE marker Cdx2. Chimera forming efficiency was calculated as number of chimeras/number of total aggregates made. Right panel: Representative images of aggregation chimeras at E4.5. We observed a number of cells in TE positions in chimeras. These cells ranged from live-looking to apoptotic, however none expressed Cdx2 and thus were not considered as viable, integrated contributions to the TE lineage.

Article Snippet: Cells (mESCs and TSCs) were first stained for surface markers CDCP1 (R&D Systems AF4515) and CD40 (BD Biosciences 562846) using antibodies at 1:100 dilutions and assayed using flow cytometry (BD LSRFortessa).

Techniques: Gene Expression, Quantitative RT-PCR, In Silico, Immunostaining, Cell Culture, Flow Cytometry, Fluorescence, RNA Sequencing, Stable Transfection, Comparison, In Vivo, Marker

Journal: bioRxiv

Article Title: Modeling signaling-dependent pluripotent cell states with boolean logic can predict cell fate transitions

doi: 10.1101/115683

Figure Lengend Snippet: (a) In silico subpopulation analysis of possible signaling input combinations with 2iL and 2i-L. The threshold values for predicted expression levels of lineage specifiers in each SCC are set as follows: Oct4=0.3, EpiTFs=0.2, and Gata6=0.5 and Cdx2=0.7. (b) Frequency of Cdx2+ population including TE-like sub-population (Cdx2+/Oct4-) after five days in culture measured by immuno-staining. Data represents the means of six replicates consisting of two biological replicates with three technical replicates in each. The representative density plot of immuno-staining of Cdx2 on day 5 is shown in the left panel. (c) Histograms for expression of TE-enriched cell surface markers, CD40 and CDCP1. Shown are mESCs treated in 2iJ (1 st column), 2i-L (2 nd column), or 2iL(3 rd column), in unsupplemented medium (1 st row) or BMP4 and ALKi supplemented medium (+B-A) (2 nd row). Control mESCs (kept in control LS) and TSCs are shown at the bottom. (d) In vivo lineage contribution frequency and chimera efficiency of H2B-Tomato ESCs treated with either 2iL or 2iJ in the presence of serum.

Article Snippet: Cells (mESCs and TSCs) were first stained for surface markers CDCP1 (R&D Systems AF4515) and CD40 (BD Biosciences 562846) using antibodies at 1:100 dilutions and assayed using flow cytometry (BD LSRFortessa).

Techniques: In Silico, Expressing, Immunostaining, Control, In Vivo

Journal: bioRxiv

Article Title: Modeling Post-Gastrula Development via Bidirectional Pluripotent Stem Cells

doi: 10.1101/2025.06.28.662107

Figure Lengend Snippet: (A) Schematic of BPSC (derived from EPSC), ESC, and EPSC differentiated spontaneously without inducing factors for 2 days. (B) Typical brightfield images of BPSC (derived from EPSC), ESC, and EPSC after two days of spontaneous differentiation without inducing factors. (C) Immunofluorescence staining of BPSC (derived from EPSC), ESC, and EPSC differentiated spontaneously for 2 days. Staining for OCT4, CDX2 and GATA6. Scale bar, 50 μm. (D) Representative FACS analysis of the BPSC (derived from EPSC), ESC, and EPSC differentiated spontaneously for 2 days. (E) UMAP plot of scRNA-seq data from E4.5 embryo cells, BPSC (derived from EPSC) after 1 or 2 days of spontaneous differentiation, and EPSC after 2 days of spontaneous differentiation. Cells were colored according their cell types and split by their origins. (F) Schematic of BPSC differentiation induced with TSM for 2-3 days. After 3 days of induction, CDX2-mCherry-positive or CDCP1-positive cells were collected by FACS and cultured with TSM. After 2 days of induction, chimeras were performed with cells injected into embryos at 8-cell stage. (G) Immunofluorescence staining of BPSC-TSC (derived from EPSC), BPSC-TSC (derived from ESC) and TSCs (derived from embryos). Staining for SOX2 and TFAP2C. Scale bar, 20 μm. (H) Immunofluorescence staining of E6.5 chimeric embryo produced using the above method . Staining for TFAP2C and GFP. Scale bar, 50 μm. BPSC and ESC are labeled by GFP. Enlarged view of the blue dotted box (right panel). (I) Immunofluorescence staining of chimeric placentas at E12.5 stage. Staining for TPBPA and GFP. Scale bar, 500 μm.

Article Snippet: For the induced differentiation of BPSC BPSC differentiation with TSM with 25μg/mL FGF4 and 1μg/mL Heparin for 3 days and collected CDX2-mCherry positive or CDCP1 (R&D Systems AF4515-SP) positive cells with FACS.

Techniques: Derivative Assay, Immunofluorescence, Staining, Cell Culture, Injection, Produced, Labeling

Journal: bioRxiv

Article Title: Modeling Post-Gastrula Development via Bidirectional Pluripotent Stem Cells

doi: 10.1101/2025.06.28.662107

Figure Lengend Snippet: (A) Representative FACS analysis of the percentages of CDCP1 positive cells in EPSC, ESC, TSC, BPSC(EPSC)-TSMD3 (EPSC-derived BPSC induced with TSM for 3 days) and BPSC(ESC)-TSMD3 (ESC-derived BPSCs induced with TSM for 3 days) (B) Immunofluorescence staining of BPSC-TSC (EPSC), BPSC-TSC (ESC) and TSC (derived from embryo). Staining for SOX2 and CDX2. Scale bar, 20 μm. (C) Representative morphological images of E6.5 chimeric embryo produced using the above method . (D) E6.5 Chimera embryo formation ratio of differentiated BPSC induced with TSM for 2 days and ESC. (E) Representative image of ESC and BPSC-diff-D2 chimera at E12.5 stage.

Article Snippet: For the induced differentiation of BPSC BPSC differentiation with TSM with 25μg/mL FGF4 and 1μg/mL Heparin for 3 days and collected CDX2-mCherry positive or CDCP1 (R&D Systems AF4515-SP) positive cells with FACS.

Techniques: Derivative Assay, Immunofluorescence, Staining, Produced

Journal: PLoS Biology

Article Title: Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

doi: 10.1371/journal.pbio.3000425

Figure Lengend Snippet: (A) DKs5, DKO3, HK2-10, and HKe3 cells were persistently treated with 5-FU (5 μM) and analyzed for CDCP1 expression by flow cytometry at the indicated time points. Dead cells were detected by 7AAD staining. The experiments were independently repeated three times in triplicate. (B) BALB/c-nu mice bearing CRC108 or CRC102 epidermal xenografts received FOLFOX treatment twice weekly for 2 weeks. Representative images (left panels) and quantification (right panel) of CDCP1+ cell frequency in untreated tumors (week 0; n = 3) and tumor remnants after FOLFOX (week 2; n = 3) were evaluated by IHC. (C) Comparison of immunohistochemical CDCP1 staining from 15 CRC patients before and after neoadjuvant therapy. All patients achieved a partial response (P). (D) CRC108 cells were FACS-sorted according to their CDCP1 expression, and the isolated subfractions were cultured as adherent monolayers overnight. After subsequent 5-FU treatment (5 μM) for 7 days, the frequencies of CDCP1+ and CDCP1– cells were again quantified by flow cytometry. Dead cells were detected by 7AAD staining. The experiments were independently repeated three times in triplicate. (E–F) Kaplan–Meier graphs showing the fraction of patients with lung-recurrence–free survival (E) or overall survival (F) for the patients with mu Kras CRCs, dichotomized by CDCP1 expression status of primary tumors. P -values are determined by the log-rank test. (G) CDCP1 expression in primary mu Kras CRCs prior to treatment versus RCB. Sample size is indicated in parentheses. Values shown are mean ± SD. A two-tailed unpaired t test was used to compare experimental groups. ** p < 0.05. Bars: 50 μm. Underlying data are available in . CDCP1, CUB-domain–containing protein 1; CRC, colorectal carcinoma; CSC, cancer stem cell; FACS, fluorescence-activated cell sorting; FOLFOX, folinic acid + fluorouracil + oxaliplatin; IHC, immunohistochemistry; Kras , Kirsten rat sarcoma viral oncogene homolog; mu Kras , mutant Kras ; RCB, residual cancer burden; 5-FU, 5-fluoropyrimidine; 7AAD, 7-aminoactinomycin.

Article Snippet: Tumor cell suspension was rinsed with PBS after detaching and centrifuged at 1,100 rpm for 10 min. Pelleted cells were resuspended in PBS with a 1:100 dilution of the anti-CDCP1 monoclonal antibody (TA502228; Origene, Rockville, MD, USA).

Techniques: Expressing, Flow Cytometry, Staining, Comparison, Immunohistochemical staining, Isolation, Cell Culture, Two Tailed Test, Fluorescence, FACS, Immunohistochemistry, Mutagenesis

Journal: PLoS Biology

Article Title: Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

doi: 10.1371/journal.pbio.3000425

Figure Lengend Snippet: (A) Design for lineage tracing experiments. FCT mice ( n = 55) received 5 mg tamoxifen 48 h prior to FOLFOX administration, after which tumor regression and relapse were monitored for 90 days. (B) The frequency of tdTomato+ cells also positive for CDCP1 post-tamoxifen injection ( n = 3 per time point). (C) Quantification of tdTomato labeling of tumor cells following tamoxifen injection ( n = 3 per time point). (D) Representative images of tumor labeling with tdTomato at day 2, 16, and 65 post-tamoxifen injection. (E) Experimental design. NOG mice were randomized to receive FOLFOX ( n = 5) or vehicle treatment ( n = 5) twice weekly for two weeks when GFP-labeled CRC101 orthotopic xenografts reached approximately 250 units under intravital imaging. Mice received 3 CidU injections (at 2 h interval) on day 1 after FOLFOX or vehicle treatment. Five days later, mice received IdU 2 h before being killed. (F) Representative images (left panels) and quantification (right panel) of a CDCP1+ CidU+ cell labeled with IdU in tumor sections from . (G) Representative markers for proliferation were examined at the mRNA level in CDCP1+ and CDCP1– subsets from DKs5 and HK2-10 cells. The experiments were independently repeated three times in triplicate. (H) Cell-cycle distribution of CD133+ CDCP1+, CD133+ CDCP1–, or CD133– fractions from mu Kras and wt Kras CRC cells was determined by combined staining with Hoechst33342 and pyronin Y. The experiments were independently repeated three times in triplicate. (I) Design for EdU label-chase experiments. NOG mice carrying CRC102 or CRC108 orthotopic xenografts ( n = 30 per group) were exposed to EdU (0.82 mg/ml in drinking water) for 8 days, followed by a 40-day chase. (J) The frequency of EdU+ cells in CD133+CDCP1+ cells, as well as in CD133+CDCP1– and CD133– subsets, was quantified throughout the label and chase phases ( n = 3 per time point). Values shown are mean ± SD. A two-tailed unpaired t test was used to compare experimental groups. ** p < 0.05. Bar: 50 μm. Underlying data are available in . CD, cluster of differentiation; CDCP1, CUB-domain–containing protein 1; CidU, 5-Chloro-2′-deoxyuridine; CRC, colorectal carcinoma; CSC, cancer stem cell; EdU, 5-ethynyl-2′-deoxyuridine; FCT, Fabpl 4X@132 -rtTA-3×(IRES-Apc . 3374) : tet-Kras G12D : CDCP1-creERT2 : Rosa26 CAG-loxP-stop-loxP-tdTomato ; FOLFOX, folinic acid + fluorouracil + oxaliplatin; GFP, green fluorescent protein; IdU, 5-Iodo-2′-deoxyuridine; Ki67, marker of proliferation Ki-67; Kras , Kirsten rat sarcoma viral oncogene homolog; MCM2, minichromosome maintenance complex component 2; mu Kras , mutant Kras ; NOG, nonobese diabetic (NOD)/Shi-scid Il2rg null ; PCNA, proliferating cell nuclear antigen; td, tandem dimer; wt Kras , wild-type Kras .

Article Snippet: Tumor cell suspension was rinsed with PBS after detaching and centrifuged at 1,100 rpm for 10 min. Pelleted cells were resuspended in PBS with a 1:100 dilution of the anti-CDCP1 monoclonal antibody (TA502228; Origene, Rockville, MD, USA).

Techniques: Injection, Labeling, Imaging, Staining, Two Tailed Test, Marker, Mutagenesis

Journal: PLoS Biology

Article Title: Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

doi: 10.1371/journal.pbio.3000425

Figure Lengend Snippet: (A) ROS levels and the ratios of [NADPH/NAPD+], [GSH/GSSG] were compared between CDCP1+ and CDCP1– fractions from mu Kras and wt Kras CRC cell lines. (B) Relative intracellular metabolite levels in DKs5-derived CDCP1+ and CDCP1– cells. (C) PPP activity of CDCP1+ and CDCP1– fractions isolated from DKs5 and HK2-10 cells. PPP activity was calculated as the difference between the rate of [1- 14 C]-glucose and [6- 14 C]-glucose oxidation to [ 14 C]-CO 2 ( n = 3). (D) Relative intracellular metabolite levels in DKs5-derived CDCP1+ cells transfected with control shRNA or shRNA targeting G6PD, TK, TA, or both TK and TA. (E) Ratio of fluxes entering directly from taken up glucose into oxidative PPP (f direct ) or after one or multiple cycles through oxidative PPP and entering back into oxidative PPP via upper glycolysis (f cycling ). The ratios were calculated using the m + 0 fraction of 6PG in HK2-10–derived CDCP1+ and CDCP1– cells cultured in media supplemented with [1- 13 C]-glucose. (F) HK2-10–derived CDCP1+ cells with G6PD or TK/TA KD were treated with or without antioxidant agent NAC (1 mM), together with or without 5-FU (5 μM) or oxaliplatin (1 μM). ROS levels (upper panel) and apoptotic rate (lower panel) were measured at 24 h and 72 h after treatment, respectively. All experiments were independently repeated three times in triplicate. Values shown are mean ± SD. A two-tailed unpaired t test was used to compare experimental groups. ** p < 0.05. Underlying data are available in . CDCP1, CUB-domain–containing protein 1; CRC, colorectal carcinoma; CSC, cancer stem cell; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; GAP, glyceraldehyde 3-phosphate; GSH, glutathione; GSSG, glutathione disulfide; G6PD, glucose-6-phosphate dehydrogenase; KD, knockdown; Kras , Kirsten rat sarcoma viral oncogene homolog; mu Kras , mutant Kras ; NAC, N-acetylcysteine; PPP, pentose phosphate pathway; ROS, reactive oxygen species; R5P, ribose 5-phosphate; shRNA, short hairpin RNA; S7P, sedoheptulose 7-phosphate; TA, transaldolase; TK, transketolase; wt Kras , wild-type Kras ; 1,3PG, 1,3-bisphosphoglycerate; 2/3PG, 2/3-phosphoglycerate; 5PRA, 5-phosphoribosylamine; 5-FU, 5-fluoropyrimidine; 6PG, 6-phosphogluconate; 6PGL, 6-phosphoglucono-D-lactone.

Article Snippet: Tumor cell suspension was rinsed with PBS after detaching and centrifuged at 1,100 rpm for 10 min. Pelleted cells were resuspended in PBS with a 1:100 dilution of the anti-CDCP1 monoclonal antibody (TA502228; Origene, Rockville, MD, USA).

Techniques: Derivative Assay, Activity Assay, Isolation, Transfection, Control, shRNA, Cell Culture, Two Tailed Test, Knockdown, Mutagenesis

Journal: PLoS Biology

Article Title: Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

doi: 10.1371/journal.pbio.3000425

Figure Lengend Snippet: (A) Co-IP of Sirt5 and TPI in CDCP1– and CDCP1+ fractions from mu Kras and wt Kras CRC cells. (B–C) DKs5 and HK2-10–derived CDCP1+ cells were treated with Au-H3K9Su for 48 h, after which lysine modifications (B) and enzyme activity (C) of endogenous TPI were determined. (D–E) DKs5 and HK2-10–derived CDCP1+ cells were transfected with Sirt5 shRNA or HA-H158Y mutant, after which lysine malonylation (D) and enzyme activity (E) of endogenous TPI were determined. (F–G) The indicated Flag-tagged TPI vectors were transfected into DKs5-derived CDCP1+ cells with stable Sirt5 KD or H158Y overexpression. WT and mutant TPI proteins were purified by Flag beads and eluted with Flag peptide, followed by determination of their lysine malonylation (F) and enzyme activity (G). All experiments were independently repeated three times in triplicate. Values shown are mean ± SD. A two-tailed unpaired t test was used to compare experimental groups. ** p < 0.05. Underlying data are available in . acetyl-K, lysine acetylation; CD, cluster of differentiation; CDCP1, CUB-domain–containing protein 1; CRC, colorectal carcinoma; glu-K, lysine glutarylation; HA, hemagglutinin; IP, immunoprecipitation; KD, knockdown; Kras , Kirsten rat sarcoma viral oncogene homolog; mal-K, lysine malonylation; mu Kras , mutant Kras ; shRNA, short hairpin RNA; Sirt5, silent mating type information regulation 2 homolog 5; succ-K, lysine succinylation; TPI, triosephosphate isomerase; WB, western blot; WT, wild type; wt Kras , wild-type Kras .

Article Snippet: Tumor cell suspension was rinsed with PBS after detaching and centrifuged at 1,100 rpm for 10 min. Pelleted cells were resuspended in PBS with a 1:100 dilution of the anti-CDCP1 monoclonal antibody (TA502228; Origene, Rockville, MD, USA).

Techniques: Co-Immunoprecipitation Assay, Derivative Assay, Activity Assay, Transfection, shRNA, Mutagenesis, Over Expression, Purification, Two Tailed Test, Immunoprecipitation, Knockdown, Western Blot

Journal: PLoS Biology

Article Title: Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

doi: 10.1371/journal.pbio.3000425

Figure Lengend Snippet: (A) The WT TPI or TPI K56E vector was transfected into DKs5 and HK2-10–derived CDCP1+ cells with stable TPI KD, after which PPP activity, percentage of PPP flux, and the ratio of [NADPH/NADP+] were determined. (B) HK2-10–derived CDCP1+ cells with TPI KD were transfected with the WT TPI or TPI K56E vector, together with or without 5-FU (5 μM) or oxaliplatin (1 μM) treatment. The apoptotic rate was measured at 72 h after treatment. (C) The frequency of CDCP1+ cells between FOLFOX and FOLFOX + Dox groups in was determined by flow cytometry ( n = 3 per time point). (D) RFS was compared between FOLFOX and FOLFOX + Dox groups in by Kaplan–Meier survival analyses. (E) Upper panels: representative lung sections from mice with recurrent tumors in showing GFP-positive lung metastases; lower panel: percentage of mice with visible lung metastases ( n = 10 per group). P -value is determined by the log-rank test. Sample size is indicated in parentheses. Throughout, values shown are mean ± SD. P -values were calculated by two-tailed t test unless otherwise indicated. ** p < 0.05. Underlying data are available in . CDCP1, CUB-domain–containing protein 1; CSC, cancer stem cell; Dox, doxycycline; FOLFOX, folinic acid + fluorouracil + oxaliplatin; GFP, green fluorescent protein; KD, knockdown; PPP, pentose phosphate pathway; shRNA, short hairpin RNA; TPI, triosephosphate isomerase; WT, wild type; 5-FU, 5-fluoropyrimidine.

Article Snippet: Tumor cell suspension was rinsed with PBS after detaching and centrifuged at 1,100 rpm for 10 min. Pelleted cells were resuspended in PBS with a 1:100 dilution of the anti-CDCP1 monoclonal antibody (TA502228; Origene, Rockville, MD, USA).

Techniques: Plasmid Preparation, Transfection, Derivative Assay, Activity Assay, Flow Cytometry, Two Tailed Test, Knockdown, shRNA

Journal: PLoS Biology

Article Title: Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

doi: 10.1371/journal.pbio.3000425

Figure Lengend Snippet: (A) Malonylation of K56 (red) induces the formation of hydrogen bonds with D88 (pink) and Q91 (blue), stabilizing the dimerization of TPI. (B) Close-up view of the interactions at the TPI dimer interface with K56 (Protein Data Bank: 4POC). Four out of the eight contacts dependent on K56 malonylation are indicated with a dashed line. The other four hydrogen bonds are the equivalent companions on the reverse side of the protein and are not visible in the picture. (C) Flag-tagged TPI or K56A/K56E mutants were each expressed in HK2-10–derived CDCP1+ cells coexpressing HA-tagged TPI. The interaction between Flag-tagged and HA-tagged proteins was determined by western blot. (D) Flag-tagged TPI or K56A/K56E mutants were each expressed in HEK293T cells, followed by treatments with or without 0.025% glutaraldehyde. The formation of TPI monomer and dimer was determined by western blot. (E) Flag-tagged TPI or K56E mutant was each expressed in HEK293T cells coexpressing HA-tagged TPI or K56E mutant, respectively. Cells were then transfected with or without myc-Sirt5, and the interaction between Flag-tagged and HA-tagged proteins was determined by western blot. CDCP1, CUB-domain–containing protein 1; HA, hemagglutinin; HEK, human embryonic kidney; IP, immunoprecipitation; MW, molecular weight; Sirt5, silent mating type information regulation 2 homolog 5; TPI, triosephosphate isomerase; WB, western blot; WT, wild type.

Article Snippet: Tumor cell suspension was rinsed with PBS after detaching and centrifuged at 1,100 rpm for 10 min. Pelleted cells were resuspended in PBS with a 1:100 dilution of the anti-CDCP1 monoclonal antibody (TA502228; Origene, Rockville, MD, USA).

Techniques: Derivative Assay, Western Blot, Mutagenesis, Transfection, Immunoprecipitation, Molecular Weight

Journal: PLoS Biology

Article Title: Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

doi: 10.1371/journal.pbio.3000425

Figure Lengend Snippet: (A) Western blot analysis of CDCP1 cleavage and plasmin secretion in mu Kras and wt Kras CRCs. (B) Western blot analysis of CDCP1 cleavage in HKe3-derived CDCP1+ cells overexpressing Flag plasminogen. (C) HKe3-10–derived CDCP1+ cells were transfected with the Flag-plasminogen vector, together with or without 5-FU (5 μM) or oxaliplatin (1 μM) treatment. The apoptotic rate was measured at 48 h after drug treatment. (D) CRC108-derived CDCP1+ cells were transfected with siRNA targeting Kras , together with or without the HA-AktCA or Flag-MEKCA mutant, following which CDCP1 cleavage and plasmin expression were analyzed by western blot. (E) Analysis of CDCP1 cleavage in CRC108-derived CDCP1+ cells pretreated with 20 μg/ml CDCP1 mAb 10-D7 or control IgG for 24 h. (F) CRC108-derived CDCP1+ cells were pretreated with 20 μg/ml 10-D7 and/or HA-PKCδCA mutant for 48 h prior to assessment of Sirt5–TPI association and TPI K56 malonylation. (G) cCDCP1 expression in CRC108-derived CDCP1– cells overexpressing Flag-cCDCP1. (H) CRC108-derived CDCP1− cells transfected with Flag-cCDCP1 were treated with or without rottlerin (2.5 μM) for 48 h prior to assessment of Sirt5–TPI association and TPI K56 malonylation. Underlying data are available in . Akt, RAC-alpha serine/threonine-protein kinase; AktCA, catalytically active mutant of Akt; cCDCP1, cleaved CDCP1; CDCP1, CUB-domain–containing protein 1; CRC, colorectal carcinoma; fCDCP1, full-length CDCP1; HA, hemagglutinin; IgG, immunoglobulin G; IP, immunoprecipitation; Kras , Kirsten rat sarcoma viral oncogene homolog; mAb, monoclonal antibody; malTPI, malonylated TPI; MEK, mitogen-activated protein kinase; MEKCA, catalytically active mutant of MEK; mu Kras , mutant Kras ; MW, molecular weight; PKCδ, protein kinase C-delta; PKCδCA, catalytically active mutant of PKCδ; Si-KRAS, KRAS siRNA; siRNA, small interfering RNA; Sirt5, silent mating type information regulation 2 homolog 5; TPI, triosephosphate isomerase; WB, western blot; wt Kras , wild-type Kras ; 5-FU, 5-fluorpyrimidine.

Article Snippet: Tumor cell suspension was rinsed with PBS after detaching and centrifuged at 1,100 rpm for 10 min. Pelleted cells were resuspended in PBS with a 1:100 dilution of the anti-CDCP1 monoclonal antibody (TA502228; Origene, Rockville, MD, USA).

Techniques: Western Blot, Derivative Assay, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Control, Immunoprecipitation, Molecular Weight, Small Interfering RNA

Journal: JCI insight

Article Title: CDCP1 regulates retinal pigmented epithelial barrier integrity for the development of experimental autoimmune uveitis.

doi: 10.1172/jci.insight.157038

Figure Lengend Snippet: Figure 1. mAb clones 2E10 and 9A2 recognize both mouse and human CDCP1. Clone 2E10 (top row) and clone 9A2 (bottom row) at a concentration of 2 μg/ mL selectively labeled mouse PYMT and human MDA 468 WT but not CDCP1-KO/KD cells, respectively. Red lines denote isotype controls; blue lines denote anti-CDCP1 IgGs. Experiments were repeated 3 times. Representative images are presented.

Article Snippet: CDCP1-KO mice were immunized with a recombinant mouse CDCP1 protein (R&D Systems), and hybridomas were generated from the immunized mice following an established protocol (32).

Techniques: Clone Assay, Concentration Assay, Labeling

Journal: JCI insight

Article Title: CDCP1 regulates retinal pigmented epithelial barrier integrity for the development of experimental autoimmune uveitis.

doi: 10.1172/jci.insight.157038

Figure Lengend Snippet: Figure 2. CDCP1 expression and regulation on mouse and human retinal pigment epithelial cells in the retina. (A) Retinal tissues and retinal pigment epithelial (RPE) cells were isolated from WT and CDCP1-KO mice, and cell lysates were prepared. CDCP1 was detected by Western blotting with a poly- clonal Ab. Here, CDCP1 was detectable in the retina and purified RPE cells from WT but not CDCP1-KO mice. Actin blots were used as loading controls. (B) Immunofluorescent staining with the anti-CDCP1 mAb clone 9A2 showed selective CDCP1 expression (red) on RPE cells in mouse and human retinas. DAPI (green) was used for nuclear staining. Scale bars: 10 μm (low-magnification images); 1 μm (high-magnification images). (C) CDCP1 expression was detected on human aRPE-19 and hTERT-RPE cells by flow cytometry with both our mAb clone 9A2 and a commercial anti-human CDCP1 mAb, clone CUB1. Gray shades indicate isotype controls; black shades indicate anti-CDCP1 IgGs. (D) CDCP1 expression on RPE cells was upregulated by treatment with IFN-γ but not IL-17 or TNF-α. RPE-19 cells were cultured with 1,000 U each of IFN-γ, IL-17, or TNF-α for 48 hours, after which CDCP1 expression was detected using flow cytometry. (E) CDCP1 expression on RPE was upregulated in vivo in EAU, as detected by immunofluorescent staining of the RPE flat mounts from WT naive and EAU mice, and using the CDCP1-KO mouse tissue as the negative control. 3D reconstruction and the orthogonal view analyses together with ezrin staining confirmed the surface expression pattern of CDCP1 on the RPE cells. CDCP1 levels (MFI) were quantitated from 4 random areas using Fiji software. Scale bars: 50 μm. Each experiment was repeated at least 3 times.

Article Snippet: CDCP1-KO mice were immunized with a recombinant mouse CDCP1 protein (R&D Systems), and hybridomas were generated from the immunized mice following an established protocol (32).

Techniques: Expressing, Isolation, Western Blot, Purification, Staining, Flow Cytometry, Cell Culture, In Vivo, Negative Control, Software

Journal: JCI insight

Article Title: CDCP1 regulates retinal pigmented epithelial barrier integrity for the development of experimental autoimmune uveitis.

doi: 10.1172/jci.insight.157038

Figure Lengend Snippet: Figure 3. CDCP1-KO mice on a C57BL/6 background develop significantly attenuated EAU after the adoptive transfer of uveitogenic T cells. (A) WT and CDCP1-KO mice were adoptively transferred with identical numbers of preactivated uveitogenic WT T cells. CDCP1-KO mice showed decreased clinical scores. n = 9 mice for each group. Mean ± SEM; *P < 0.05, 2-way ANOVA. (B) Topical endoscopy fundus imaging (TEFI) revealed retinal damages (arrows and black-dot-outlined area) and vasculitis resulting in white sheathing (white-dot-outlined area) caused by EAU in WT mice but not in CDCP1-KO mice. (C and D) Confocal scanning laser ophthalmoscopy (cSLO) and spectral-domain optical coherence tomography (SD-OCT) revealed more hyperreflective foci (arrows) and lesions (black-dot-outlined area) in WT mice compared with KO mice. Images were taken from all the mice. Representative images are presented. Reflective particles in the vitreous in SD-OCT images were quantified using ImageJ software. Reduced numbers and sizes of particles were observed in KO mice. Each dot represents a mouse. Mean ± SEM; *P < 0.05, **P < 0.01, t test. (E) Histopathological imaging revealed significantly greater cell infiltration in the vitreous and folds in the retinas of WT mice relative to KO mice and decreased histopathological scores in the latter. Each dot rep- resents a mouse. Mean ± SEM; *P < 0.05, t test. Scale bars: 100 μm.

Article Snippet: CDCP1-KO mice were immunized with a recombinant mouse CDCP1 protein (R&D Systems), and hybridomas were generated from the immunized mice following an established protocol (32).

Techniques: Adoptive Transfer Assay, Imaging, Tomography, Software

Journal: JCI insight

Article Title: CDCP1 regulates retinal pigmented epithelial barrier integrity for the development of experimental autoimmune uveitis.

doi: 10.1172/jci.insight.157038

Figure Lengend Snippet: Figure 4. CDCP1-KO mice on DBA/1 background develop significantly attenuated EAU after the adoptive transfer of uveitogenic T cells. (A) WT and CDCP1-KO mice were adoptively transferred with identical numbers of preactivated uveitogenic WT T cells. CDCP1-KO mice showed decreased clinical scores. n = 5 mice for each group. Mean ± SEM; *P < 0.05, 2-way ANOVA. (B and C) Histopathological imaging revealed (B) greater cell infiltration in the vitreous and folds in the retinas of WT mice relative to KO mice and (C) decreased histopathological scores in the latter. Each dot represents a mouse. Mean ± SEM; *P < 0.05, t test. Scale bars: 100 μm. (D) Confocal scanning laser ophthalmoscopy (cSLO) and spectral-domain optical coherence tomog- raphy (SD-OCT) revealed more hyperreflective foci (arrows), lesions (black-dot-outlined area), and swollen retinal vessels (red-dot-outlined area) in WT mice than in KO mice. Images were taken from all the mice. Representative images are presented.

Article Snippet: CDCP1-KO mice were immunized with a recombinant mouse CDCP1 protein (R&D Systems), and hybridomas were generated from the immunized mice following an established protocol (32).

Techniques: Adoptive Transfer Assay, Imaging

Journal: JCI insight

Article Title: CDCP1 regulates retinal pigmented epithelial barrier integrity for the development of experimental autoimmune uveitis.

doi: 10.1172/jci.insight.157038

Figure Lengend Snippet: Figure 5. RPE tight junctions are severely disrupted in WT but not CDCP1-KO mice during EAU development. WT and CDCP1-KO mice were adoptively transferred with identical numbers of preactivated uveitogenic WT T cells, and 6 days later, mice were perfused and RPE flat mounts were prepared and stained for ZO-1 and CD3. (A) Confocal analyses of the samples showed massive numbers of lesions and RPE tight junction disruptions in WT mice and few lesions and relatively intact tight junctions in the CDCP1-KO mouse RPE flat mounts. Scale bars: 50 μm. (B) Further closer analyses also identified infiltrat- ing CD3+ T cells (green) only in some of the lesions in the RPE flat mounts from the WT mice. IMARIS 3D reconstruction of ZO-1 and CD3 in WT EAU after background subtraction and surface module rebuild. Scale bars: 20 μm. (C and D) Representative horizontal orientation showing CD3+ T cells migrating through the retina layer. Representative images are presented. Lesions are identified by dotted lines and arrows point to the CD3+ T cells. Scale bars: 20 μm.

Article Snippet: CDCP1-KO mice were immunized with a recombinant mouse CDCP1 protein (R&D Systems), and hybridomas were generated from the immunized mice following an established protocol (32).

Techniques: Staining

Journal: JCI insight

Article Title: CDCP1 regulates retinal pigmented epithelial barrier integrity for the development of experimental autoimmune uveitis.

doi: 10.1172/jci.insight.157038

Figure Lengend Snippet: Figure 6. CDCP1 knockdown on RPE cells impairs T cell transmigration by reducing CD6-stimulated cytoskeletal remodeling and tight junction disrup- tion. (A) Flow cytometry analysis showed decreased CDCP1 levels on CDCP1-knockdown (CDCP1-KD) RPE cells. (B) WT and CDCP1-KD RPE cells were cul- tured in top chambers of Transwell inserts to form monolayers for T cell migration assays. CFSE-labeled mouse T cells were activated and overlaid on RPE monolayers. After 18 hours, numbers of T cells in the top and bottom chambers were quantified using flow cytometry, and the percentages of migration were calculated. Experiments were repeated 3 times. **P < 0.01, t test. (C) After CD6 stimulation, permeabilities of WT and CDCP1-KD RPE monolayers were measured using a FITC-dextran leakage assay. CDCP1-KO RPE monolayer had decreased barrier permeability. Experiments were repeated twice. *P < 0.05, 2-way ANOVA. (D) WT and CDCP1-KD RPE cells were analyzed by confocal microscopy after a 6-hour incubation with hCD6-Fc or human IgG protein. Representative confocal images of ZO-1 (green) and DAPI (magenta). White arrowheads marked ZO-1 loss. (E) CDCP1-KD RPE cells have increased ZO-1 expression compared with WT cells after CD6 treatment. (F) Representative images of actin filaments (green) and activated myosin light chain (pMLC, red) in WT and CDCP1-KD RPE cells after a 4-hour incubation with 1 μg/mL recombinant hCD6-Fc fusion protein or human IgG. (G and H) CD6 treatment induced robust actin filament bundle and stress fiber formation but had a milder effect on the CDCP1-KD cells, as shown by mean fluorescence intensity (MFI). Data were quantitated and analyzed using 2-way ANOVA and Tukey’s test after repeating the experiment 3 times (n = 6 images/condition, experi- ments were repeated twice) and are presented as the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 1 μm.

Article Snippet: CDCP1-KO mice were immunized with a recombinant mouse CDCP1 protein (R&D Systems), and hybridomas were generated from the immunized mice following an established protocol (32).

Techniques: Knockdown, Transmigration Assay, Flow Cytometry, Migration, Labeling, Permeability, Confocal Microscopy, Incubation, Expressing, Recombinant, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Metastatic prostate cancer‐derived extracellular vesicles facilitate osteoclastogenesis by transferring the CDCP1 protein

doi: 10.1002/jev2.12312

Figure Lengend Snippet: Screening of molecules expressed on EVs that promote osteoclastogenesis. (a) Schematic representation of the proteome analysis of PC3M‐ and PNT2‐derived EVs. (b) Venn diagram of the number of detected proteins in PC3M‐ and PNT2‐derived EVs. (c) Analysis of LC‒MS/MS data by spectral count in PC3M‐ and PNT2‐derived EVs. Twenty‐one proteins that were significantly enriched in PC3M‐derived EVs were selected as candidate proteins to promote osteoclast differentiation (Fisher's exact test; p < 0.003). (d) Schematic protocol for small‐scale screening using the siRNA SMART pool in PC3M cells to detect candidate proteins that promote osteoclast differentiation. (e) Flow diagram of proteins used for selecting the candidate protein CDCP1. (f) EVs from PC3M and PNT2 cells were analysed by immunoblotting using an anti‐human CDCP1 antibody. Proteins from whole cell lysates or EVs were separated on SDS‒PAGE gels, followed by immunoblotting. A 10 μg cell lysate sample was used for the detection of CDCP1 and actin. A 0.5 μg EV protein sample was used for the detection of CDCP1. (g) Immunoelectron microscopy images of PC3M EVs and PNT2 EVs labelled with anti‐CDCP1 antibody. The black dots emphasized by yellow arrows indicate the presence of CDCP1. Scale bar, 200 nm.

Article Snippet: HEK cells at 90% confluency were transfected with the pCMV6‐Myc‐DDK‐tagged ORF clone of human CDCP1 (Origene Technologies, Inc., Rockville, MD) and vector plasmid DNA in 24‐well dishes using Lipofectamine 3000 reagent in accordance with the manufacturer's instructions (Invitrogen).

Techniques: Derivative Assay, Western Blot, Immuno-Electron Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Metastatic prostate cancer‐derived extracellular vesicles facilitate osteoclastogenesis by transferring the CDCP1 protein

doi: 10.1002/jev2.12312

Figure Lengend Snippet: CDCP1 located on EVs is a key molecule for osteoclastogenesis. (a) Cell lysates from HEK293 cells overexpressing CDCP1 or the control were analysed by immunoblotting using anti‐CDCP1 and anti‐actin antibodies. Actin was used as the loading control. (b) EVs from HEK293 cells overexpressing CDCP1 or the control were analysed by immunoblotting using anti‐CDCP1 and anti‐CD9 antibodies. CD9 was used as the loading control. (c) Immunoelectron microscopy images of HEK293 cell‐derived EVs labelled with anti‐CDCP1 antibody. The black dots emphasized by yellow arrows indicate the presence of CDCP1. Scale bar, 200 nm. (d) Osteoclast precursor cells were cultured with EVs derived from HEK293 control cells (control) or CDCP1‐overexpressing HEK293 (CDCP1‐OE)‐derived EVs (30 μg/mL) in the presence of RANKL (10 ng/mL). At the end of Day 4, osteoclast precursor‐induced cells were fixed and stained for TRAP, and the number of TRAP‐positive multinucleated cells containing more than 3 nuclei (MNCs) was counted. (e) Quantitative PCR analysis was performed for markers of osteoclast differentiation, including TRAP and CTSK. Error bars represent s.d. of triplicate experiments. ** indicates a significant difference from the RANKL‐stimulated group (R10) as determined by Dunnett's test (* p < 0.05, ** p < 0.01). R10 represents 10 ng/mL RANKL.

Article Snippet: HEK cells at 90% confluency were transfected with the pCMV6‐Myc‐DDK‐tagged ORF clone of human CDCP1 (Origene Technologies, Inc., Rockville, MD) and vector plasmid DNA in 24‐well dishes using Lipofectamine 3000 reagent in accordance with the manufacturer's instructions (Invitrogen).

Techniques: Control, Western Blot, Immuno-Electron Microscopy, Derivative Assay, Cell Culture, Staining, Real-time Polymerase Chain Reaction

Journal: Journal of Extracellular Vesicles

Article Title: Metastatic prostate cancer‐derived extracellular vesicles facilitate osteoclastogenesis by transferring the CDCP1 protein

doi: 10.1002/jev2.12312

Figure Lengend Snippet: The CDCP1 protein is detected in EVs derived from metastatic prostate cancer patients. (a) Immunoblotting for CDCP1 protein expression levels in EVs derived from nonmetastatic and bone metastatic prostate cancer patients. Each lane was loaded with EVs from 100 μL plasma. (b) The results of bone scintigraphy of representative cases. (c) Quantification of the relative expression levels of CDCP1 protein normalized to CD9 in EVs.

Article Snippet: HEK cells at 90% confluency were transfected with the pCMV6‐Myc‐DDK‐tagged ORF clone of human CDCP1 (Origene Technologies, Inc., Rockville, MD) and vector plasmid DNA in 24‐well dishes using Lipofectamine 3000 reagent in accordance with the manufacturer's instructions (Invitrogen).

Techniques: Derivative Assay, Western Blot, Expressing, Clinical Proteomics

Journal: Molecular cancer

Article Title: Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling.

doi: 10.1186/s12943-015-0415-0

Figure Lengend Snippet: Fig. 1 Phenotypic screening in primary NSCLC tumour cells. a Images of cells derived from NSCLC primary tumour #1 cultured in three different conditions. b Effects of the scFv-Fc antibody panel upon NSCLC tumour #1 cell growth in three culture conditions, measured by Cell-Titre Glo (CTG) luminescence signal. Each antibody was individually dosed (without normalising concentrations) and cells were grown in 96-well plates. Positive (anti-IGF1R) and negative control antibodies were dosed in multiple replicates on each plate to establish consistency between plates. Each data point indicates a single well. Black horizontal bars indicate the average value for a sample class. c Scatter plot comparing the effects of scFv-Fc antibodies on NSCLC tumour #1 cell growth grown in spheroids and in standard monolayer cultures. Each data point indicates a single antibody (or replicate of the controls). The dashed box indicates a group of antibodies that strongly inhibited growth of cell monolayers but not spheroids. The solid-line box indicates a group of antibodies with a weak inhibitory effect in both spheroids and monolayers. The orange-coloured datapoint represents an antibody that was later shown to bind CDCP1 (αCDCP1-Ab3 in Fig. 2)

Article Snippet: Matriptase digest of recombinant human CDCP1 in the presence of antibodies 50 nM recombinant human CDCP1 extracellular domain with a C-terminal FLAG-His10 tag was treated with 5 nM recombinant matriptase catalytic domain (R + D Systems, cat# 3946-SE) in the presence of 200 nM anti-CDCP1 antibodies or 100 nM aprotinin protease inhibitor.

Techniques: Derivative Assay, Cell Culture, Negative Control