Additional file 1 of Single-cell N6-methyladenosine-related genes function within the tumor microenvironment to affect the prognosis and treatment sensitivity in patients with gastric cancer

Supplementary Material 1: Fig. S1. Myeloid cells in the TME. (A) t-SNE plot of 13 myeloid cell subclusters in the TME. (B) t-SNE plot of 7 myeloid cell subtypes in the TME. (C) Marker genes of each myeloid cell type. (D) Expression level of marker genes in 7 myeloid cell subtypes. (E) t-SNE plot of MRG score in 7 myeloid cell subtype

Expression of CTTN in ESCC and normal esophageal tissues.

<p>Note:*<i>P</i><0.005.</p

Genome-Wide Gene Expression Profile Analyses Identify CTTN as a Potential Prognostic Marker in Esophageal Cancer

<div><p>Aim</p><p>Esophageal squamous cell carcinoma (ESCC) is one of the most common fatal malignances of the digestive tract. Its prognosis is poor mainly due to the lack of reliable markers for early detection and prognostic prediction. Here we aim to identify the molecules involved in ESCC carcinogenesis and those as potential markers for prognosis and as new molecular therapeutic targets.</p><p>Methods</p><p>We performed genome-wide gene expression profile analyses of 10 primary ESCCs and their adjacent normal tissues by cDNA microarrays representing 47,000 transcripts and variants. Candidate genes were then validated by semi quantitative reverse transcription-PCR (RT-PCR), tissue microarrays (TMAs) and immunohistochemistry (IHC) staining.</p><p>Results</p><p>Using an arbitrary cutoff line of signal log ratio of ≥1.5 or ≤−1.5, we observed 549 up-regulated genes and 766 down-regulated genes in ESCCs compared with normal esophageal tissues. The functions of 302 differentially expressed genes were associated with cell metabolism, cell adhesion and immune response. Several candidate deregulated genes including four overexpressed (CTTN, DMRT2, MCM10 and SCYA26) and two underexpressed (HMGCS2 and SORBS2) were subsequently verified, which can be served as biomarkers for ESCC. Moreover, overexpression of cortactin (CTTN) was observed in 126/198 (63.6%) of ESCC cases and was significantly associated with lymph node metastasis (P = 0.000), pathologic stage (P = 0.000) and poor survival (P<0.001) of ESCC patients. Furthermore, a significant correlation between CTTN overexpression and shorter disease-specific survival rate was found in different subgroups of ESCC patient stratified by the pathologic stage (P<0.05).</p><p>Conclusion</p><p>Our data provide valuable information for establishing molecules as candidates for prognostic and/or as therapeutic targets.</p></div

Kaplan-Meier plots for the Disease-specific survival rate of ESCC patients.

<p>(A) Kaplan-Meier plots for the Disease-specific survival (DSS) rate of ESCC patients with (n = 126, green line) or without (n = 72, blue line) CTNN overexpression. (B) Kaplan-Meier plots for the DSS rate of ESCC patients with pathologic stage I+IIA (n = 113, blue line) or IIB+III (n = 85, green line).</p

The differential expression of CTNN in ESCC.

<p>(A) Semiquantitative reverse transcription-PCR (RT-PCR) was applied to compare expression status of CTTN, DMRT2, MCM10, SCYA26, HMGCS2 and SORBS2 between 8 pairs of primary ESCC tumor samples and matched normal esophageal epithelia. GAPDH was used as an internal control. (B) Representative of CTNN expression in a pair of ESCC (right) and adjacent normal tissue (left) detected by immunostaining with anti-CTNN antibody (brown). The slide was counterstained with hematoxylin (Original magnification: upper ×100, bottom ×200).</p

Kaplan-Meier plots for the DSS rate in ESCC patients subgrouped into pathologic stage I-IIA (A) and pathologic stage IIB-III (B) as differentiated by with (+) or without (−)-CTTN overexpression.

<p>Kaplan-Meier plots for the DSS rate in ESCC patients subgrouped into pathologic stage I-IIA (A) and pathologic stage IIB-III (B) as differentiated by with (+) or without (−)-CTTN overexpression.</p

Association between CTTN over-expression and clinicopathologic characteristics of patients with ESCC (n = 198).

<p>*<i>P</i><0.05.</p

Additional file 1 of PHF5A promotes esophageal squamous cell carcinoma progression via stabilizing VEGFA

Supplementary Material

Additional file 1 of Long non-coding RNA NEAT1 mediated RPRD1B stability facilitates fatty acid metabolism and lymph node metastasis via c-Jun/c-Fos/SREBP1 axis in gastric cancer

Additional file 1: Supplementary Table 1. List of antibodies used in this project. Supplementary Table 2. List of PCR primers for expression and cloning. Supplementary Table 3. List of PCR primers for Luciferase assay. Supplementary Table 4. List of PCR primers for ChIP assay. Supplementary Table 5. List of probes for EMSA assay. Supplementary Table 6. Association of RPRD1B upregulation with clinicopathologic features in 191 GCs. Supplementary Fig. 1. (A) RT–qPCR showing that RPRD1B was the most significantly overexpressed gene in 8 target genes in both GC tissue and metastatic lymph node (n = 10). (B, C) Levels of the RPRD1B and Mettl3 proteins and mRNAs after Mettl3 knockdown or overexpression in HGC27 cells. (D) RIP-qPCR showing the enrichment of m6A in HGC27 cells after Mettl3 depletion, independent Student’s t test. (E) The decay rate of the RPRD1B mRNA after treatment with 2.5 μM actinomycin D for the indicated times in AGS cells with Mettl3 knockdown or overexpression. GAPDH was served as the loading control. Data are presented as the means ± SD of three independent experiments. (*, P < 0.05; ***, P < 0.001). Supplementary Fig. 2. (A, B) Wound-healing assay showing that RPRD1B overexpression promoted the migration of SGC7901 cells and RPRD1B knockdown inhibited the migration of BGC823 cells at 0, 24, and 48 h after scratch wounding. (C) Transwell migration assay showing that SR11302 inhibited the RPRD1B-induced migration of SGC7901 cells. Scale bar, 200 μm. (D) Transwell migration assay showed that NEAT1 was upregulated in RPRD1B-overexpressing HGC27 and SGC7901 cells. The effect was diminished by SR11302. (E) NEAT1 was downregulated in AGS and BGC823 RPRD1B-silenced cells and rescued by c-Jun and c-Fos. Scale bar, 20 μm. Data are presented as the means ± SD of three independent experiments. (NS, not significant; **, P < 0.01; ***, P < 0.001). Supplementary Fig. 3. (A) CoIP assay validated that hnRNPA2B1, not YTHDF1, directly interacted with NEAT1 in HGC27 cells. (B) Levels of the hnRNPA2B1 and RPRD1B proteins and mRNAs after RPRD1B inhibition in RPRD1B-overexpressing SGC7901 cells. The results are summarized as the means ± SD of three independent experiments. (C) MeRIP-qPCR showing the enrichment of m6A in HGC27 cells after hnRNPA2B1 depletion. (D) The decay rate of the RPRD1B mRNA after treatment with 2.5 μM actinomycin D for the indicated times following hnRNPA2B1 knockdown in RPRD1B-overexpressing HGC27 cells. (E) RIP-qPCR showing the enrichment of hnRNPA2B1 on the RPRD1B mRNA in RPRD1B-overexpressing HGC27 cells with NEAT1 silencing. (F) The IHC staining and Oil red O staining were performed in 10 cases of GC cohort with RPRD1B overexpression. The IHC score were summarized. GAPDH was served as the loading control. Data are presented as the means ± SD of three independent experiments. (**, P < 0.01; ***, P < 0.001). Supplementary Fig. 4. Preliminary IHC staining showed the optimum concentration of antibody and verified the correctness and specificity of antibody