mouse cripto biotinylated ab  (R&D Systems)

Journal: Frontiers in Oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: CRIPTO (CR-1) is dynamically expressed in stem cell-enriched cultures of adenocarcinoma (AC) and squamous cell carcinoma (SCC). (A) Hematoxylin/Eosin staining of paraffin-embedded xenograft sections obtained from two SCC and two AC spheroid cultures (upper panels) and of the parental patient tumor (lower panels), 20X magnification, scale bar 50 μm. Pictures in the upper panels are representative of xenograft sections derived from three different mice. (B) Flow cytometry analysis of surface CRIPTO expression on two SCC and two AC spheroid cultures derived from patients represented in (A) and in . (C) Immunofluorescence staining for CRIPTO in combination with E-Cadherin or Prolyl 4-Hydroxylase subunit beta (P4HB) to show respectively plasmamembrane and endoplasmic reticulum localization on AC1 cells, 60X magnification, 1.8X zoom, scale bar 10 μm. (D) Values obtained from flow cytometry analysis of surface CRIPTO (indicated as percentage of positive cells) in AC1 (light purple rhombus) and SCC1 (light blue square) at the indicated days. The histogram represents the percentage of FACS-positive cells for 7-amino actinomycin D (7-AAD). FACS plots are shown in Supplemental Figure 1 .

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Staining, Derivative Assay, Flow Cytometry, Expressing, Immunofluorescence

Journal: Frontiers in Oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: CRIPTO (CR-1) expression regulates cells proliferation and stem cell gene expression in NSCLC spheroids. (A) Immunoblot analysis of CRIPTO and βActin in SCC1 spheroids transduced with the empty vector (Vector), with CRIPTO shRNA vector (CRIPTO KO) or with exogenous CRIPTO (CRIPTO-over). (B) CRIPTO and stem cell genes (OCT3/4, SOX2, NANOG) mRNA expression in SCC1 transduced with vector, CRIPTO KO or CRIPTO-over sequences. (C) ATP assay performed on SCC1 transduced with the vector, CRIPTO KO or CRIPTO-over sequences, performed 4 days after transduction (D) . Cell cycle analysis of SCC1 transduced as above, performed 3 days after transduction. (E) Soft agar pictures (left; two technical replicates for each sample) and graph (right) of colony forming assay performed on control (Vector), CRIPTO KO and CRIPTO overexpressing SCC1 spheroids. The results are evaluated as colony formation in semisolid culture and expressed as normalized colony size/percentage over plated cells. (F) ELISA assay performed on SCC1 cells transduced with empty vector, CRIPTO KO sequences and CRIPTO overexpression vector. (G) Invasion/migration assay performed on vector-transduced, CRIPTO KO and CRIPTO overexpressing SCC1 cells. *P < 0.05; **P < 0.01, ***P < 0.001 by unpaired student’s t test (transduced vs vector).

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Expressing, Gene Expression, Western Blot, Transduction, Plasmid Preparation, shRNA, ATP Assay, Cell Cycle Assay, Control, Enzyme-linked Immunosorbent Assay, Over Expression, Migration

Journal: Frontiers in Oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: NSCLC subpopulations expressing high or low CRIPTO (CR-1) levels are interconvertible in vitro and in vivo . (A) FACS-based separation of CRIPTO high and CRIPTO low subpopulations of AC1 spheroid cultures. (B) Self-renewal capacity of sorted CRIPTO high and CRIPTO low AC1 spheroid cells, evaluated as colony formation in semisolid culture and expressed in bottom panels as normalized colony size/percentage over plated cells. (C) qRT-PCR analysis of CRIPTO and stem cell related genes (OCT3/4, SOX2, NANOG) performed on CRIPTO high and CRIPTO low subpopulations of AC1 spheroids. (D) Proliferation curve of CRIPTO high (dark pink square) and CRIPTO low (light blue dot) subpopulations or Bulk (burgundy triangle) cultures of AC1 spheroids, starting at day 0 (after sorting) and monitored at the indicated times. (E) qRT-PCR analysis of CRIPTO high (light purple histogram) and CRIPTO low (light blue histogram) subpopulations at the indicated times starting at day 0 after sorting. (F) Left: In vivo growth of tumor xenografts obtained by subcutaneous inoculation of CRIPTO high (dark pink square) and CRIPTO low (light blue dot) subpopulations or Bulk (burgundy triangle) cultures of AC1 spheroids, monitored at the indicated times. Results shown are the mean ± SEM of values obtained using 4 mice per group. *P < 0.05; **P < 0.01, ***P < 0.001. Middle: representative confocal images of CRIPTO (red) staining of CRIPTO high , CRIPTO low and Bulk tumors. 40X magnification, scale bar 50 μm. Right: quantification of CRIPTO performed on 3 sets composed of 5 fields/group. **P < 0.01. AU, arbitrary units.

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Expressing, In Vitro, In Vivo, Quantitative RT-PCR, Staining

Journal: Frontiers in Oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: Chemotherapy treatment increases CRIPTO (CR-1) expression and tumor progression in NSCLC xenografts. (A) Flow cytometry analysis of CRIPTO on SCC1 cells treated with vehicle only (Vehicle) or with chemotherapeutic agents (Cis+Gem early and Cis+Gem late). Cells in the Cis+Gem samples were treated with Cisplatin 5 μM plus Gemcitabine 25 μM for 4 days then washed, replated and analyzed after 3 additional days (Cis+Gem early) or 7 additional days (Cis+Gem late). The graph shows the mean ± SD of two independent experiments. Ns, non-significant, ***P < 0.001. (B) Left: Xenograft volume of SCC1 spheroids cells treated with Vehicle (Vehicle, light blue square) or with Cisplatin plus Gemcitabine (Cis+Gem, Dark pink dots). Mean ± SEM, 6 mice/group. **P < 0.01 (two-tailed t test). Middle: representative confocal images of CRIPTO (red) and TUNEL (green) staining of Vehicle and Cis+Gem treated tumors. 40X magnification, scale bar 50 μm. Right: quantification of CRIPTO and TUNEL performed on 3 sets composed of 5 fields/group. **P < 0.01. AU, arbitrary units. (C) Tumor variation after treatment withdrawal of SCC1 spheroids treated with vehicle (Vehicle, light blue square), and Cis plus Gemcitabine (Cis+Gem, dark pink dots). Mean ± SEM, 4 mice/group. (D) qRT-PCR analysis of CRIPTO and Cyclin E mRNA expression of SCC1-derived xenografts, monitored during treatment and after treatment withdrawal at the indicated times. *P < 0.05; **P < 0.01, ***P < 0.001.

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Expressing, Flow Cytometry, Two Tailed Test, TUNEL Assay, Staining, Quantitative RT-PCR, Derivative Assay