cd133 apc mab  (R&D Systems)

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: PDPN+ glioma-stem-like cells form aggressive tumors in vivo and mark a stem-like radioresistant subpopulation of cells. (A) PDPN protein is expressed in seven of the nine GSC lines shown. (B) Flow cytometry of GSCs shows PDPN expression is pervasive and more prominent in these GSCs. (C) Kaplan–Meier survival analysis of GSC11 PDPN−/CD133+, PDPN+/CD133+, and PDPN+/CD133− FACS-sorted subpopulations orthotopically injected into the brains of immunocompromised mice reveal that mice harboring tumors from CD133+/PDPN+ cells had a median survival of 99 days (n=5), while mice that received CD133+/PDPN− cells did not succumb to tumor formation (n=3) (p=0.0136, log-rank test). (D) PDPN+ FACS-sorted subpopulations have a higher sphere formation ability (*p<0.005, multiple t-tests, PDPN+ vs . PDPN− groups). (E) PDPN+ FACS-sorted subpopulations have a higher surviving fraction of cells after 2 Gy of radiation (*p<0.005, multiple t-tests, PDPN+ vs . PDPN− groups). (F) Transcriptome analysis revealed distinct gene expression signatures in PDPN+ and PDPN− sorted GSCs (three different cell lines). (G) GSEA of differentially expressed genes (p<0.05) demonstrated significant enrichment of the mesenchymal subtype signature (NES=2.04, q=0.02) in PDPN+ populations.

Article Snippet: FACSAria (BD Biosciences) was used to analyze and sort GSCs based on PDPN and CD133 expression using a PE-conjugated mAb to PDPN (clone NZ-1, AngioBio) alone or in combination with an APC-conjugated mAb to CD133 (clone 293C3, Miltenyi Biotech).

Techniques: In Vivo, Flow Cytometry, Expressing, Injection, Gene Expression

Journal: American Journal of Cancer Research

Article Title: Arsenic trioxide suppresses lung adenocarcinoma stem cell stemness by inhibiting m6A modification to promote ferroptosis

doi:

Figure Lengend Snippet: Inhibition of proliferation and m6A formation by ATO in LASCs from A549 cells. (A) Tumorsphere formation in A549 cells induced by treatment with the sphere formation medium. Tumor sphere formation after treatment for 1, 3, 5, and 7 days was evaluated using the sphere formation assay. (B) Increase in CD133-positive cells in A549 cells treated with sphere formation medium. The percentages of CD133+ cells were measured by flow cytometry. (C) Suppression of LASCs cell viability by ATO treatment for 24 or 48 h. LASCs from A549 cells were treated with 0, 0.625, 1.25, 2.5, 5, 10, 20, or 40 mM of ATO, followed by detection of cell viability by the CCK-8 method. (D and E) Effects of ATO treatment on the expression of m6A regulator genes in LASCs from A549 cells. The mRNA (D) and protein (E) levels of major m6A writers and erasers in LASCs from A549 cells were analyzed by quantitative RT-PCR and western blotting, respectively. (F) Decrease in total m6A content in LASCs from A549 cells induced by ATO treatment. The total m6A levels in LASCs from A549 cells were determined using the dot blot method. ATO: arsenic trioxide; LASCs: lung adenocarcinoma stem cells; NC: negative control; METTL14/16: methyltransferase-like protein 14/16; WTAP: Wilms’ tumor 1-associating protein; ZC3H13: zinc finger CCCH domain-containing protein 13; FTO: fat mass and obesity-associated gene; ALKBH5: alkylated DNA repair protein alkB homolog 5; *P < 0.05.

Article Snippet: In brief, approximately 10 6 LASCs were first blocked in 100 μl of blocking buffer (PBS solution containing 2% BSA and 1% FBS) for 15 min in darkness, which were then incubated in darkness with diluted antibodies, including CD133 APC Mab (#FAB11331A-100; R&D Systems) and APC Mouse IgG2a Isotype Control (#E-AB-F09802E; Elabscience Biotechnology) for 30 min at 4°C.

Techniques: Inhibition, Tube Formation Assay, Flow Cytometry, CCK-8 Assay, Expressing, Quantitative RT-PCR, Western Blot, Dot Blot, Negative Control, Wilms Tumor Assay

Journal: American Journal of Cancer Research

Article Title: Arsenic trioxide suppresses lung adenocarcinoma stem cell stemness by inhibiting m6A modification to promote ferroptosis

doi:

Figure Lengend Snippet: Modulation of LASCs from A549 cells ferroptosis and stemness by ATO. (A) Effects of ATO treatment on the sphere-forming capacities of LASCs. LASCs were treated with 20 mM of ATO for 48 h, and the capacity of tumorsphere formation was assessed by the tumorsphere formation assay. (B) Decreased percentage of CD133-positive LASCs caused by ATO treatment. The percentages of CD133+ cells were analyzed using the flow cytometry method. (C) Promotion of ROS production in LASCs induced by ATO treatment. LASCs were treated with 20 mM of ATO for 24 h, and the ROS levels in LASCs were quantitated by fluorescence microscopy. (D) Increases in iron ion levels in LASCs after ATO treatment. The iron ion contents in the LASCs were detected by the colorimetric assay. (E and F) Inhibition of GPX4 gene expression by ATO treatment in LASCs. The mRNA (E) and protein (F) levels of GPX4 in LASCs were detected by quantitative RT-PCR and western blotting, respectively, after ATO treatment at 20 mM for 24 or 48 h. (G) Morphological aberrations in LASCs mitochondria induced by ATO treatment. The morphological features of mitochondria in LASCs were observed via transmission electron microscopy. LASCs: lung adenocarcinoma stem cells; NC: negative control; ATO: arsenic trioxide; GPX4: glutathione peroxidase 4; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *P < 0.05.

Article Snippet: In brief, approximately 10 6 LASCs were first blocked in 100 μl of blocking buffer (PBS solution containing 2% BSA and 1% FBS) for 15 min in darkness, which were then incubated in darkness with diluted antibodies, including CD133 APC Mab (#FAB11331A-100; R&D Systems) and APC Mouse IgG2a Isotype Control (#E-AB-F09802E; Elabscience Biotechnology) for 30 min at 4°C.

Techniques: Tube Formation Assay, Flow Cytometry, Fluorescence, Microscopy, Colorimetric Assay, Inhibition, Gene Expression, Quantitative RT-PCR, Western Blot, Transmission Assay, Electron Microscopy, Negative Control

Journal: American Journal of Cancer Research

Article Title: Arsenic trioxide suppresses lung adenocarcinoma stem cell stemness by inhibiting m6A modification to promote ferroptosis

doi:

Figure Lengend Snippet: Effects of ZC3H13 overexpression and Fer-1 on the stemness of LASCs from A549 cells. A. ZC3H13 mRNA levels in LASCs that overexpress the ZC3H13 gene or treated with Fer-1. ZC3H13 mRNA levels in LASCs were analyzed by quantitative PCR. B. ZC3H13 protein abundances in LASCs with ZC3H13 gene overexpression or under the treatment. Western blotting was performed to assess ZC3H13 protein levels. C. Total m6A level changes in LASCs under ATO treatment combined with ZC3H13 overexpression or Fer-1 treatment. m6A levels in LASCs were detected via the dot blot method. D. Promotion of the sphere formation capacity of LASCs under ATO treatment by ZC3H13 overexpression or Fer-1 treatment. The tumorsphere-forming capacity of LASCs was assessed by the sphere formation assay. E. Recovery of the percentages of CD133-positive LASCs under ATO treatment by ZC3H13 gene overexpression or Fer-1 treatment. Percentages of CD133+ LASCs cells were quantitated by flow cytometry. Fer-1: ferrostatin-1; ZC3H13: zinc finger CCCH domain-containing protein 13; LASCs: lung adenocarcinoma stem cells; NC: negative control; ATO: arsenic trioxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *P < 0.05.

Article Snippet: In brief, approximately 10 6 LASCs were first blocked in 100 μl of blocking buffer (PBS solution containing 2% BSA and 1% FBS) for 15 min in darkness, which were then incubated in darkness with diluted antibodies, including CD133 APC Mab (#FAB11331A-100; R&D Systems) and APC Mouse IgG2a Isotype Control (#E-AB-F09802E; Elabscience Biotechnology) for 30 min at 4°C.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Dot Blot, Tube Formation Assay, Flow Cytometry, Negative Control

Journal: American Journal of Cancer Research

Article Title: Arsenic trioxide suppresses lung adenocarcinoma stem cell stemness by inhibiting m6A modification to promote ferroptosis

doi:

Figure Lengend Snippet: Abrogation of si-ZC3H13-induced ferroptosis enhancement and stemness inhibition in LASCs from A549 cells by Fer-1 treatment. (A and B) Expression levels of ZC3H13 gene in LASCs treated with si-ZC3H13-1 (or si-ZC3H13-2) and Fer-1. ZC3H13 mRNA (A) and protein (B) levels in LASCs were quantitated by quantitative RT-PCR and western blotting, respectively. (C) Changes in total m6A modification levels in LASCs treated with a combination of si-ZC3H13-1 (or si-ZC3H13-2) and Fer-1. Total m6A levels in LASCs were analyzed using the dot blot assay. (D) Effects of Fer-1 treatment on ROS content in LASCs transfected with si-ZC3H13-1 (or si-ZC3H13-2). Fluorescence microscopy was used for the quantitation of ROS content in LASCs. (E) Iron ion level downregulation in LASCs treated with si-ZC3H13-1 (or si-ZC3H13-2) by Fer-1 treatment. The iron ion contents in LASCs were compared using the colorimetric method. (F and G) Promotion of GPX4 gene expression by Fer-1 treatment in LASCs with ZC3H13 knockdown by si-ZC3H13-1 (or si-ZC3H13-2). The mRNA (F) and protein (G) levels of the GPX4 gene in LASCs were detected by quantitative PCR and western blotting, respectively. (H) Modulation of mitochondrial functions by Fer-1 treatment in LASCs with silenced ZC3H13 expression. Morphological alterations of mitochondria in LASCs were assessed by transmission electron microscopy. (I) Recovery of the sphere-forming capacity of ZX3H13-silenced LASCs by Fer-1 treatment. Tumor sphere formation by LASCs was evaluated using the sphere formation assay. (J) Elevation of CD133-positive cell percentages in ZX3H13-silenced LASCs by Fer-1 treatment. Flow cytometry was performed to quantify CD133-positive LASCs percentages. ZC3H13: zinc finger CCCH domain-containing protein 13; LASCs: lung adenocarcinoma stem cells; NC: negative control; si-ZC3H13-1: siRNA fragment 1 of ZC3H13; si-ZC3H13-2: siRNA fragment 2 of ZC3H13; Fer-1: Ferrostatin-1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPX4: glutathione peroxidase 4; *P < 0.05.

Article Snippet: In brief, approximately 10 6 LASCs were first blocked in 100 μl of blocking buffer (PBS solution containing 2% BSA and 1% FBS) for 15 min in darkness, which were then incubated in darkness with diluted antibodies, including CD133 APC Mab (#FAB11331A-100; R&D Systems) and APC Mouse IgG2a Isotype Control (#E-AB-F09802E; Elabscience Biotechnology) for 30 min at 4°C.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Modification, Dot Blot, Transfection, Fluorescence, Microscopy, Quantitation Assay, Gene Expression, Knockdown, Real-time Polymerase Chain Reaction, Transmission Assay, Electron Microscopy, Tube Formation Assay, Flow Cytometry, Negative Control

Journal: American Journal of Cancer Research

Article Title: Arsenic trioxide suppresses lung adenocarcinoma stem cell stemness by inhibiting m6A modification to promote ferroptosis

doi:

Figure Lengend Snippet: Effects of interference with ZC3H13 on the stemness of LASCs from A549 cells. A. ZC3H13 mRNA levels in LASCs with ZC3H13 combined with ATO treatment. ZC3H13 mRNA levels in LASCs were analyzed by quantitative PCR. B. ZC3H13 protein abundances in LASCs with ZC3H13 combined with ATO treatment. Western blotting was performed to evaluate ZC3H13 protein levels. C. Alterations of total m6A levels in LASCs interference with ZC3H13 combined with ATO treatment. m6A levels in LASCs were detected via the dot blot method. D. Promotion of the sphere formation capacity of LASCs interference with ZC3H13 combined with ATO treatment. The tumorsphere-forming capacity of LASCs was evaluated using the sphere formation assay. E. Percentages of CD133-positive LASCs after interference with ZC3H13 combined with ATO treatment. Percentages of CD133+ LASCs cells were quantitated by flow cytometry. ZC3H13: zinc finger CCCH domain-containing protein 13; LASCs: lung adenocarcinoma stem cells; NC: negative control; si-ZC3H13-1: siRNA fragment 1 of ZC3H13; si-ZC3H13-2: siRNA fragment 2 of ZC3H13; ATO: arsenic trioxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *P < 0.05.

Article Snippet: In brief, approximately 10 6 LASCs were first blocked in 100 μl of blocking buffer (PBS solution containing 2% BSA and 1% FBS) for 15 min in darkness, which were then incubated in darkness with diluted antibodies, including CD133 APC Mab (#FAB11331A-100; R&D Systems) and APC Mouse IgG2a Isotype Control (#E-AB-F09802E; Elabscience Biotechnology) for 30 min at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Dot Blot, Tube Formation Assay, Flow Cytometry, Negative Control

Journal: American Journal of Cancer Research

Article Title: Arsenic trioxide suppresses lung adenocarcinoma stem cell stemness by inhibiting m6A modification to promote ferroptosis

doi:

Figure Lengend Snippet: ATO impaired LASCs tumorigenicity by inhibiting ZC3H13 to promote ferroptosis. (A-C) Effect of ZC3H13 overexpression on the volumes and weights of tumors formed in ATO-treated naked mice. The volume (A and B) and weights (C) of tumors formed in naked mice were measured weekly after LASCs from A549 cells injection, which lasted for four consecutive weeks. (D and E) Alterations in ZC3H13 gene expression in tumors derived from ZC3H13-overexpressing LASCs in naked mice treated with ATO. ZC3H13 mRNA and protein levels in tumors were detected by quantitative PCR and western blotting, respectively. (F) Elevation of total m6A content in tumors developed from ZC3H13-overexpressing LASCs in naked mice treated with ATO Total m6A levels in tumor tissues were analyzed using the dot bot. (G) Changes in CD133+ cells in tumors derived from ZC3H13-overexpressing LASCs in naked mice treated with ATO. Immunofluorescence was performed to assess CD133+ cells in mice tissues. (H) ROS contents in tumor tissues developed from ZC3H13-overexpressing LASCs in naked mice under ATO treatment. (I) Influence of ZC3H13 overexpression on the iron ion level of tumor tissues in mice treated with ATO. Iron ion levels in tumor tissues were measured using the colorimetric method. (J and K) ZC3H13 overexpression-induced alterations of GPX4 expression in tumors developed from LASCs in naked mice. GPX4 mRNA and protein levels in tumor tissues were measured by quantitative PCR and western blotting, respectively. LASCs: lung adenocarcinoma stem cells; NC: negative control; ATO: arsenic trioxide; ZC3H13: zinc finger CCCH domain-containing protein 13; GPX4: glutathione peroxidase 4; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *P < 0.05.

Article Snippet: In brief, approximately 10 6 LASCs were first blocked in 100 μl of blocking buffer (PBS solution containing 2% BSA and 1% FBS) for 15 min in darkness, which were then incubated in darkness with diluted antibodies, including CD133 APC Mab (#FAB11331A-100; R&D Systems) and APC Mouse IgG2a Isotype Control (#E-AB-F09802E; Elabscience Biotechnology) for 30 min at 4°C.

Techniques: Over Expression, Injection, Gene Expression, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Expressing, Negative Control