Journal: Frontiers in Immunology

Article Title: HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway

doi: 10.3389/fimmu.2023.1258074

Figure Lengend Snippet: HOXA3 regulated the differentiation, migration and proliferation of 3PPE. (A) Schematic representation of siRNA transduction during the differentiation of 3PPE from hESCs. (B) The expression of HOXA3 gene was assessed by qRT-PCR after the treatment with HOXA3 siRNA. (C, D) Western blot was used to evaluate the protein expression of HOXA3 in cells treated with HOXA3 siRNA (C) , and protein levels were normalized to those of housekeeping gene GAPDH (D) . (E) Heatmap showed the transcriptional changes of key genes in 3PPE after the treatment with HOXA3 siRNA. (F) The expression changes of BMP4, SIX1, TBX1, PAX9, PBX1 were determined by qRT-PCR in cells treated with HOXA3 siRNA. (G, H) Western blot was used to evaluate the protein expressions of PAX9, SIX1 and BMP4 in cells treated with HOXA3 siRNA (C) , and protein levels were normalized to those of housekeeping gene GAPDH (D) . (I) Representative confocal images of immunostainings of SIX1 in cells treated with HOXA3 siRNA, and DAPI was used to stain the nucleus (blue). Scale bar = 50 µm. (J) The immunofluorescent quantifications of staining for SIX1 protein (J) in (I) . (K) The differentially expressed genes under HOXA3 inhibition showed biological process by GO function enrichment analysis after the treatment with HOXA3 siRNA. (L) GSEA analysis showed that the genes negatively regulating epithelial cell migration were down-regulated after HOXA3 inhibition with a normalized enrichment score of 1.250 (FDR q value of 0.417). (M) GSEA analysis showed that the genes regulating epithelial cell proliferation were down-regulated after HOXA3 inhibition with a normalized enrichment score of 1.256 (FDR q value of 0.902). (N) Representative images (Left panel) and quantitation (Right panel) of wound-healing assay for the measurement of cell migration 24 hours after the treatment with HOXA3 siRNA. (O) Heatmap exhibited transcriptional changes of genes associated with cell migration in 3PPE after the treatment with HOXA3 siRNA. (P) The expression changes of CTHRC1 and INSL3 were assessed by qRT-PCR in cells treated with HOXA3 siRNA. (Q) Cell proliferation assay was determined by CKK8 at 24, 48 and 72 hours after the treatment of cells with HOXA3 siRNA. (R) The expression changes of proliferation related genes MCM2, CDC45L and c-Myc was assessed by qRT-PCR 48 hours after the treatment of cells with HOXA3 siRNA. Values were presented in mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. qRT-PCR data were shown normalised to the NC condition. n = 3 independent biological repeats.

Article Snippet: The final lysates were incubated with 1 μg HOXA3 antibody (Santa Cruz) or 1 μg mouse immunoglobulin G (IgG) antibody (Beyotime) at 4°C overnight.

Techniques: Migration, Transduction, Expressing, Quantitative RT-PCR, Western Blot, Staining, Inhibition, Quantitation Assay, Wound Healing Assay, Proliferation Assay

Journal: Frontiers in Immunology

Article Title: HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway

doi: 10.3389/fimmu.2023.1258074

Figure Lengend Snippet: Wnt/β-catenin signaling and EPHB2 were the downstream effector of HOXA3 in 3PPE. (A) GSEA analysis showed that the genes regulating Wnt signaling pathway were down-regulated after HOXA3 inhibition with a normalized enrichment score of 1.379 (FDR q value of 0.593). (B) Heat map exhibited the transcriptional changes of genes associated with WNT signaling pathway in cells of 3PPE after the treatment with HOXA3 siRNA. (C) The expression changes of genes associated with WNT signaling pathway were assessed by qRT-PCR in cells treated with HOXA3 siRNA. (D, E) Western blot was used to evaluate the protein expressions of FZD8, GSK3β, p-GSK3β, β-Catenin, Cyclin D1 and c-Myc in cells treated with HOXA3 siRNA (D) , and the protein levels were normalized to those of housekeeping gene GAPDH (E) . (F) The expression changes of EPHB2 was assessed by qRT-PCR 48 hours after the treatment of cells with HOXA3 siRNA. (G, H) Western blot was used to evaluate the protein expression of EPHB2 in cells treated with HOXA3 siRNA (G) , and the protein levels were normalized to those of housekeeping gene GAPDH (H) . (I) Upstream binding sites of HOXA3 in the promoter of EPHB2 were predicted. Sequence logo of HOXA3 (Upper panel). Possible binding sites of HOXA3 in the location and area of EPHB2 promoter (Bottom panel). (J) PCR was performed in ChIP assays to amplify DNA samples precipitated with HOXA3 antibodies using specific primers to evaluate the recruitment of HOXA3 on EPHB2 promoter. Normal rabbit immunoglobulin G (IgG) or no antibody was used as negative controls. (K) Quantitative ChIP-qPCR assay was performed with the 3PPE cells using IgG and HOXA3 antibody followed by amplification of DNA fragments of EPHB2 promoter. Data was normalized to input fraction and the results were calibrated to that of IgG which was set 1. Values were presented in mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. qRT-PCR data were shown normalised to the NC condition. n = 3 independent biological repeats.

Article Snippet: The final lysates were incubated with 1 μg HOXA3 antibody (Santa Cruz) or 1 μg mouse immunoglobulin G (IgG) antibody (Beyotime) at 4°C overnight.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Binding Assay, Sequencing, ChIP-qPCR, Amplification

Journal: Frontiers in Immunology

Article Title: HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway

doi: 10.3389/fimmu.2023.1258074

Figure Lengend Snippet: HOXA3 regulated EPHB2-mediated Wnt signaling which in turn regulated migration and proliferation of 3PPE. (A) The expression of EPHB2 gene was assessed by qRT-PCR after the treatment with EPHB2 siRNA. (B) The expression changes of genes associated with WNT signaling pathway were assessed by qRT-PCR in cells treated with EPHB2 siRNA. (C) Cell proliferation assay was determined by CKK8 at 24, 48 and 96 hours after the treatment of cells with EPHB2 siRNA. (D) Representative images (Left panel) and quantitation (Right panel) of wound-healing assay for the measurement of cell migration 24 hours after the treatment with EPHB2 siRNA. (E) The expression change of INSL3 was assessed by qRT-PCR in cells treated with EPHB2 siRNA. (F) qRT-PCR analysis for expression of EPHB2 in 3PPE treated with Si-HOXA3, NC plus EFNB2 or Si-HOXA3 plus EFNB2. (G) qRT-PCR analysis for expression of FZD8, FZD5, TCF12, GSK3B and CNND1 in 3PPE treated with Si-HOXA3, NC plus EFNB2 or Si-HOXA3 plus EFNB2. (H) Representative images (Left panel) and quantification (Right panel) of Wound healing assays for the measurement of migration of 3PPE 24 hours after the treatment with Si-HOXA3, NC plus EFNB2 or Si-HOXA3 plus EFNB2. (I) qRT-PCR analysis for expression of INSL3 in 3PPE treated with Si-HOXA3, NC plus EFNB2 or Si-HOXA3 plus EFNB2. (J, K) Cell cycle distribution of cells was determined by flow cytometry with propidium iodide staining 48 hours after the treatment with SiHOXA3, NC plus EFNB2 or SiHOXA3 plus EFNB2. (L, M) The expression changes of cell cycle markers Cdc25c and PCNA (L) and cell proliferation markers (M) were determined by qRT-PCR in cells after the treatment with Si-HOXA3, NC plus EFNB2 or Si-HOXA3 plus EFNB2. (N) Schematic diagram illustrating that HOXA3 regulated the development of hESC-derived 3PPE. Values were presented in mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. qRT-PCR data were shown normalised to the NC condition. n = 3 independent biological repeats.

Article Snippet: The final lysates were incubated with 1 μg HOXA3 antibody (Santa Cruz) or 1 μg mouse immunoglobulin G (IgG) antibody (Beyotime) at 4°C overnight.

Techniques: Migration, Expressing, Quantitative RT-PCR, Proliferation Assay, Quantitation Assay, Wound Healing Assay, Flow Cytometry, Staining, Derivative Assay

Journal: Oncogenesis

Article Title: LncRNA HOXA-AS3 confers cisplatin resistance by interacting with HOXA3 in non-small-cell lung carcinoma cells

doi: 10.1038/s41389-019-0170-y

Figure Lengend Snippet: a qPCR analyses of the mRNA expression of HOXA3 following HOXA-AS3 suppression for 48 h. b , c Western blotting detection of the protein levels of HOXA3 following HOXA-AS3 suppression for 48 h. d RNA pull-down assay and western blotting detection of HOXA-AS3 binding to the HOXA3 protein. (L lysate load, FT flow-through; E eluate). e Mass spectrum detection of HOXA-AS3 binding to the HOXA3 protein. f RNA pull-down assay and qPCR detection of HOXA-AS3 binding to the mRNA of HOXA3. (L lysate load, FT flow-through, E eluate). g RNA immunoprecipitation detection of the enrichment of HOXA-AS3 using an anti-HOXA3 antibody. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Small interfering RNAs (siRNAs) targeting HOXA-AS3 (si-HOXA-AS3-651, si-HOXA-AS3-728, si-HOXA-AS3-3507), HOXA3 (#sc-38675, Santa Cruz Biotechnology) or negative control (NC) siRNAs were transfected into NSCLC cells using Lipofectamine 3000.

Techniques: Expressing, Western Blot, Pull Down Assay, Binding Assay, RNA Immunoprecipitation

Journal: Oncogenesis

Article Title: LncRNA HOXA-AS3 confers cisplatin resistance by interacting with HOXA3 in non-small-cell lung carcinoma cells

doi: 10.1038/s41389-019-0170-y

Figure Lengend Snippet: a – d CCK-8 assay of the viability of NSCLC cell lines following cisplatin treatment (0, 1.049, 2.097, 4.195, 8.389, 16.779 μM) under HOXA3 suppression for 48 h. e – f EDU assay of the proliferation of NSCLC cell lines under the IC 50 of cisplatin under HOXA3 suppression for 48 h. All scale bars indicate 50 μm (Green, EDU staining of dividing cells; blue, Hoechest 33342 staining of nuclei). g , h Flow cytometry detection of the apoptosis rate of NSCLC cell lines under the IC 50 of cisplatin under HOXA3 suppression for 48 h. i – m Western blotting of HOXA3, E-cadherin, and Vimentin levels following HOXA3 suppression for 48 h. n Western blotting of Twist1 levels following HOXA3 suppression for 48 h. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Small interfering RNAs (siRNAs) targeting HOXA-AS3 (si-HOXA-AS3-651, si-HOXA-AS3-728, si-HOXA-AS3-3507), HOXA3 (#sc-38675, Santa Cruz Biotechnology) or negative control (NC) siRNAs were transfected into NSCLC cells using Lipofectamine 3000.

Techniques: CCK-8 Assay, EdU Assay, Staining, Flow Cytometry, Western Blot

Journal: Oncogenesis

Article Title: LncRNA HOXA-AS3 confers cisplatin resistance by interacting with HOXA3 in non-small-cell lung carcinoma cells

doi: 10.1038/s41389-019-0170-y

Figure Lengend Snippet: a – d CCK-8 assay of the viability of NSCLC cell lines following cisplatin treatment (0, 1.049, 2.097, 4.195, 8.389, 16.779 μM) under HOXA-AS3 + HOXA3, or HOXA-AS3 only suppression for 48 h. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Small interfering RNAs (siRNAs) targeting HOXA-AS3 (si-HOXA-AS3-651, si-HOXA-AS3-728, si-HOXA-AS3-3507), HOXA3 (#sc-38675, Santa Cruz Biotechnology) or negative control (NC) siRNAs were transfected into NSCLC cells using Lipofectamine 3000.

Techniques: CCK-8 Assay

Journal: Oncogenesis

Article Title: LncRNA HOXA-AS3 confers cisplatin resistance by interacting with HOXA3 in non-small-cell lung carcinoma cells

doi: 10.1038/s41389-019-0170-y

Figure Lengend Snippet: a Growth curve of tumor volumes and b Growth curve of body weight calculated every 2 days after inoculation. c Tumor size after the tumors was harvested. d Tumor weight after the tumors was harvested. e – f Representative images of immunohistochemical detection of the proliferation index Ki-67 in vivo. g , h Representative images of immunohistochemical detection of the apoptosis index TUNEL in vivo. i , j Western blotting detection of HOXA3 in vivo. k qPCR detection of the mRNA expression levels of HOXA3 and HOXA-AS3 in vivo. (* vs cisplatin, * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Small interfering RNAs (siRNAs) targeting HOXA-AS3 (si-HOXA-AS3-651, si-HOXA-AS3-728, si-HOXA-AS3-3507), HOXA3 (#sc-38675, Santa Cruz Biotechnology) or negative control (NC) siRNAs were transfected into NSCLC cells using Lipofectamine 3000.

Techniques: Immunohistochemical staining, In Vivo, TUNEL Assay, Western Blot, Expressing