Journal: PeerJ

Article Title: Role of ELK1 in regulating colorectal cancer progression: miR-31-5p/CDIP1 axis in CRC pathogenesis

doi: 10.7717/peerj.15602

Figure Lengend Snippet: (A) The levels of miR-31-5p expression in collected human colorectal cancer tissues and adjacent normal intestinal mucosa tissues were detected by qRT-PCR. (B) The levels of miR-31- 5p expression in the human normal colon epithelial cell line, FHC, and colorectal cancer cell lines, NCI-H498, RKO, LoVo, HT29, and Coco-2, were detected by qRT-PCR. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Colorectal cancer cell lines, RKO (ATCC ® CRL-2577), LoVo (CCL-229TM), HT29 (HTB-38TM), Coco-2 (HTB-37TM), and NCI-H498 (CCL-254TM), as well as FHC cell line (CRL-1831TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: PeerJ

Article Title: Role of ELK1 in regulating colorectal cancer progression: miR-31-5p/CDIP1 axis in CRC pathogenesis

doi: 10.7717/peerj.15602

Figure Lengend Snippet: (A) miR-31-5p inhibition was achieved in RKO and LoVo cells by transfection with an miR-31-5p inhibitor. The migration abilities of RKO and LoVo cells with miR-31-5p inhibition were detected by wound healing assays (B, scale: 100 µm), and their invasion capabilities were assessed by the Transwell assay (C, 50 µm). (D, E) Electron microscopy (D) and immunoblotting (E) were used to detect autophagy. (F, G, H, I) The levels of BAX protein were detected by immunoblotting (F), and caspase 3 (G), caspase 9 (H), and caspase 11(I) activity in RKO and LoVo cells was detected by ELISA. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Colorectal cancer cell lines, RKO (ATCC ® CRL-2577), LoVo (CCL-229TM), HT29 (HTB-38TM), Coco-2 (HTB-37TM), and NCI-H498 (CCL-254TM), as well as FHC cell line (CRL-1831TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Inhibition, Transfection, Migration, Transwell Assay, Electron Microscopy, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: PeerJ

Article Title: Role of ELK1 in regulating colorectal cancer progression: miR-31-5p/CDIP1 axis in CRC pathogenesis

doi: 10.7717/peerj.15602

Figure Lengend Snippet: RKO and LoVo cells were transfected with ELK1 siRNA or miR-31-5p mimics. (A) miR-31-5p expression was detected by qRT-PCR. (B) ELK1 expression was detected by qRT-PCR. (C) Cell migration was assessed by a wound healing assay. Scale: 100 µm; (D) cell invasion was detected by the Transwell assay, Scale: 50 µm. (E, F) Electron microscopy (E) and immunoblotting (F) were used to detect autophagy. (G, H, I, J) BAX protein levels were detected by immunoblotting (G), and the levels of caspase 3(H), caspase 9 (I), and caspase 11 (J) activity in RKO and LoVo cells were detected by ELISA. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Colorectal cancer cell lines, RKO (ATCC ® CRL-2577), LoVo (CCL-229TM), HT29 (HTB-38TM), Coco-2 (HTB-37TM), and NCI-H498 (CCL-254TM), as well as FHC cell line (CRL-1831TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Migration, Wound Healing Assay, Transwell Assay, Electron Microscopy, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: PeerJ

Article Title: Role of ELK1 in regulating colorectal cancer progression: miR-31-5p/CDIP1 axis in CRC pathogenesis

doi: 10.7717/peerj.15602

Figure Lengend Snippet: (A) CDIP1 overexpression was achieved in RKO and LoVo by transfection with CDIP1 plasmids. (B, C, D) BAX protein levels detected by immunoblotting, and the levels of caspase 3, caspase 9, and caspase 11 activity in RKO and LoVo cells were detected by ELISA. (E, F) The invasion capabilities of RKO and LoVo cells with CDIP1 overexpression were detected by the Transwell assay (E, Scale: 50 µm) and their migration abilities were detected by the wound healing assay (F, Scale: 100 µm). (G, H) Electron microscopy (G) and immunoblotting (H) were used to detect autophagy. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: Colorectal cancer cell lines, RKO (ATCC ® CRL-2577), LoVo (CCL-229TM), HT29 (HTB-38TM), Coco-2 (HTB-37TM), and NCI-H498 (CCL-254TM), as well as FHC cell line (CRL-1831TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Over Expression, Transfection, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Transwell Assay, Migration, Wound Healing Assay, Electron Microscopy

Journal: PeerJ

Article Title: Role of ELK1 in regulating colorectal cancer progression: miR-31-5p/CDIP1 axis in CRC pathogenesis

doi: 10.7717/peerj.15602

Figure Lengend Snippet: (A) After being transfected with miR-31-5p mimics or CDIP1 siRNA, RKO and LoVo cells were analyzed for their miR-31-5p levels by qRT-PCR. The migration ability of transfected RKO and LoVo cells was evaluated by the wound healing assay (B, Scale: 100 µm), and their invasion capabilities were analyzed by the Transwell assay (C, Scale: 50 µm). (D, E) Electron microscopy (D) and immunoblotting (E) were used to detect autophagy. (F, G, H, I) Western blotting was used to measure Bax protein expression, and ELISA was used to detect caspase 3 (G), caspase 9 (H), and caspase 11 (I) activity in RKO and LoVo cells. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: Colorectal cancer cell lines, RKO (ATCC ® CRL-2577), LoVo (CCL-229TM), HT29 (HTB-38TM), Coco-2 (HTB-37TM), and NCI-H498 (CCL-254TM), as well as FHC cell line (CRL-1831TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Transfection, Quantitative RT-PCR, Migration, Wound Healing Assay, Transwell Assay, Electron Microscopy, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Cancer Science

Article Title: DYRK2 promotes chemosensitivity via p53‐mediated apoptosis after DNA damage in colorectal cancer

doi: 10.1111/cas.15973

Figure Lengend Snippet: DYRK2 is not involved in cell proliferation and migration under basal conditions. (A) DYRK2‐KO cells (HCT116, RKO, DLD‐1, and SW480) were generated using CRISPR‐Cas9 technology. Expression of DYRK2, phospho‐p53‐Ser46, and p53 was analyzed using immunoblotting. (B) Proliferation of parental and DYRK2‐KO cells. Cell proliferation was measured using the MTS assay. Data are presented as mean ± SEM ( n = 3). ns, not significant.

Article Snippet: Human CRC cell lines HCT116, HCT116 p53(−/−), RKO, DLD‐1, and SW480 were obtained from the JCRB Cell Bank, ATCC, and Horizon Discovery.

Techniques: Migration, Generated, CRISPR, Expressing, Western Blot, MTS Assay

Journal: Cancer Science

Article Title: DYRK2 promotes chemosensitivity via p53‐mediated apoptosis after DNA damage in colorectal cancer

doi: 10.1111/cas.15973

Figure Lengend Snippet: DYRK2‐KO decreases the chemosensitivity of p53 wild‐type CRC cells to 5‐FU and oxaliplatin. (A–D) Cell viability under 5‐FU and oxaliplatin in parental and DYRK2‐KO cells (HCT116, RKO, DLD‐1, and SW480). Cell viability was measured using the MTS assay. The IC 50 values of 5‐FU and oxaliplatin in parental and DYKR2‐KO cells. Data are presented as the mean ± SEM ( n = 3). (E, F) Colony formation assay under 5‐FU (5 μM) and oxaliplatin (5 μM) in parental and DYRK2‐KO cells. The number of colonies in each well was counted and the fold change was calculated, along with the parental cells. Data are presented as the mean ± SEM ( n = 3). (G) Expression of phospho‐p53‐Ser46, and p53 in parental and DYRK2‐KO HCT116 cells was analyzed using immunoblotting. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: Human CRC cell lines HCT116, HCT116 p53(−/−), RKO, DLD‐1, and SW480 were obtained from the JCRB Cell Bank, ATCC, and Horizon Discovery.

Techniques: MTS Assay, Colony Assay, Expressing, Western Blot

Journal: Cancer Science

Article Title: DYRK2 promotes chemosensitivity via p53‐mediated apoptosis after DNA damage in colorectal cancer

doi: 10.1111/cas.15973

Figure Lengend Snippet: DYRK2 promotes DNA damage‐induced apoptosis under 5‐FU and oxaliplatin in p53 wild‐type CRC cells. (A) Expression of phospho‐p53‐ser46, p53, cleaved PARP, and cleaved caspase‐3 under 5‐FU (5 μM) and oxaliplatin (5 μM) treatment for 72 h were analyzed using immunoblotting. (B, C) Ratio of total apoptotic parental and DYRK2‐KO HCT116 cells under 5‐FU (5 μM) and oxaliplatin (5 μM) for 48 h was analyzed using annexin V‐FITC/PI double staining. Data are presented as the mean ± SEM ( n = 3). (D) Ratio of total apoptotic parental and DYRK2‐KO RKO cells under 5‐FU (5 μM) and oxaliplatin (5 μM) for 48 h was analyzed using annexin V‐FITC/PI double staining. Data are presented as the mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01, ns, not significant.

Article Snippet: Human CRC cell lines HCT116, HCT116 p53(−/−), RKO, DLD‐1, and SW480 were obtained from the JCRB Cell Bank, ATCC, and Horizon Discovery.

Techniques: Expressing, Western Blot, Double Staining

Journal: Communications Biology

Article Title: Homodimerized cytoplasmic domain of PD-L1 regulates its complex glycosylation in living cells

doi: 10.1038/s42003-022-03845-4

Figure Lengend Snippet: a PD-L1 forms more homodimers in the Endoplasmic reticulum. HEK293T cells were transiently transfected with PD-L1-I247tag-Flag and pIRE4-Azi plasmids. 1 mM of Azi was added to the medium. Cells were treated with UV for 15 min. The ER, Golgi, and plasma membrane fractionation were isolated and separated on SDS-PAGE gels. Immunoblotting was performed using anti-Flag antibodies. b PD-L1 △C homodimerized with WT PD-L1. Azi was genetically incorporated into V76 of PD-L1-Flag or PD-L1 △C -Flag in HEK293T cells expressing PD-L1-HA. Cells were treated with UV for 20 min. Cell lysates were treated with PNGase F to remove the N-glycan. c , e PD-L1 △C suppresses complex glycosylation of WT PD-L1. c , d WT PD-L1-HA and PD-L1 △C -Flag plasmids were transfected into RKO KO PD-L1 Cells. c PD-L1 △C affects the pattern of PD-L1 that showed on immunoblotting. The anti-PD-L1 antibodies (clone number E1L3N) could recognize the full-length PD-L1, but not the PD-L1 △C . d Cell lysates for lane 3 and lane 4 were treated with PNGase F. Samples for lane 5 and lane 6 were treated with 10 µM tunicamycin (TM) for 24 h before cell collecting. e RKO KO PD-L1 cells were cotransfected with PD-L1-Flag and PD-L1 △C -Flag plasmids. Cell lysates for lanes 4–6 were treated with Endo H. Lines and arrowheads colored blue indicate full-length PD-L1. Red arrowheads indicate the PD-L1 △C proteins.

Article Snippet: HEK293T cells were purchased from National Collection of Authenticated Cell Cultures, RKO cells were purchased from iCell Bioscience Inc. All cell lines were tested negative for mycoplasma contamination.

Techniques: Transfection, Fractionation, Isolation, SDS Page, Western Blot, Expressing

Journal: Communications Biology

Article Title: Homodimerized cytoplasmic domain of PD-L1 regulates its complex glycosylation in living cells

doi: 10.1038/s42003-022-03845-4

Figure Lengend Snippet: a PD-L1-Azi interaction with PD-1 was suppressed by 3RE mutation. Azi was incorporated at V76 of WT PD-L1 or PD-L1-3RE mutant. HEK293T cells were irradiated with UV in the absence or presence of PD-1-His protein. Shown are immunoblotting using anti-Flag and anti-His antibodies. b PD-L1 △C suppressed WT PD-L1 cross-linking with PD-1. Cells expressing PD-L1-V76Azi-Flag or coexpressing PD-L1-V76Azi-Flag and PD-L1 △C -Flag were incubated with PD-1-His protein. Covalent cross-linking was induced by UV treatment for 20 min. c , d T-cell mediated tumor cell killing assay in RKO KO PD-L1 cells expressing PD-L1 and PD-L1-3RE. Green fluorescent was counted as dead cells. Representative phases are shown. Scale bar, 100 μm. d The ratio of dead cells was quantified. N = 5. e Schematic diagram of PD-L1 homodimer function. All samples were treated with PNGase F to remove the N-glycan.

Article Snippet: HEK293T cells were purchased from National Collection of Authenticated Cell Cultures, RKO cells were purchased from iCell Bioscience Inc. All cell lines were tested negative for mycoplasma contamination.

Techniques: Mutagenesis, Irradiation, Western Blot, Expressing, Incubation

Journal: Drug discovery today

Article Title: Slit/Robo pathway: a promising therapeutic target for cancer

doi: 10.1016/j.drudis.2014.09.008

Figure Lengend Snippet: Expression of Slit/Robo

Article Snippet: Colon cancer , RKO , Slit2 , ↑ , [ 85 ].

Techniques: Expressing