Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: ALDH1A1 maintains the cancer stem-like cells properties of esophageal squamous cell carcinoma by activating the AKT signal pathway and interacting with β-catenin.

doi: 10.1016/j.biopha.2020.109940

Figure Lengend Snippet: Fig. 1. Differential expression of ALDH1A1 protein in ESCC tissues and the lost ALDH1A1 expression associated with poor overall survival. IHC analysis of ALDH1A1 expression in ESCC tissues. (A) Low expression of ALDH1A1 protein in hyperplastic esophagus tissues. (B, C) Low expression of ALDH1A1 protein in ESCC. (D) High expression of ALDH1A1 protein in ESCC. Scale bar =100 μm. (E) Overall survival of ESCC patients stratified in accordance with ALDH1A1 protein expression.

Article Snippet: The primary antibodies were incubated at 4 °C overnight: anti-AKT (dilution 1:1000), anti-AKT1 (1:1000), p-AKT (Ser473) (1:1000), p-AKT (Thr308) (1:1000) and Vimentin (1:500) were purchased from CST, βcatenin (1:2000), c-Myc (1:1000) from Proteintech, ALDH1A1 (1:1000, AL: ALDH1A1; L: low expression; H: High expression; HEs: Hyperplastic esophagus tissues; ESCCs: Esophageal squamous cell carcinoma tissues.

Techniques: Quantitative Proteomics, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: ALDH1A1 maintains the cancer stem-like cells properties of esophageal squamous cell carcinoma by activating the AKT signal pathway and interacting with β-catenin.

doi: 10.1016/j.biopha.2020.109940

Figure Lengend Snippet: Fig. 2. Ectopic expression of ALDH1A1 promoted spherogenesis and colony formation of ESCC cells in vitro. (A) Tumor sphere formation assays in KYSE-510/NC, KYSE-510/ALDH1A1, KYSE-150/NC, and KYSE-150/ALDH1A1 cells. Scale bar =200 μm. (B) Spherogenesis rate. The graph is the summarized data of three in- dependent experiments, ***P < 0.001. (C) ALDH1A1 expression promoted colony formation of KYSE-150 cells. The graph is the summarized data of three in- dependent experiments, ***P < 0.001. (D) Cell survival assay showed that ALDH1A1 expression promoted TE-1 cell resistance to 5-Fu. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The primary antibodies were incubated at 4 °C overnight: anti-AKT (dilution 1:1000), anti-AKT1 (1:1000), p-AKT (Ser473) (1:1000), p-AKT (Thr308) (1:1000) and Vimentin (1:500) were purchased from CST, βcatenin (1:2000), c-Myc (1:1000) from Proteintech, ALDH1A1 (1:1000, AL: ALDH1A1; L: low expression; H: High expression; HEs: Hyperplastic esophagus tissues; ESCCs: Esophageal squamous cell carcinoma tissues.

Techniques: Expressing, In Vitro, Clonogenic Cell Survival Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: ALDH1A1 maintains the cancer stem-like cells properties of esophageal squamous cell carcinoma by activating the AKT signal pathway and interacting with β-catenin.

doi: 10.1016/j.biopha.2020.109940

Figure Lengend Snippet: Fig. 3. Ectopic expression of ALDH1A1 promoted tumor initiation in vivo. (A) Equal KYSE-150/NC and KYSE-150/ALDH1A1 cells were injected into different axillaries (white arrow) of six BALB/c nude mice. (B) Transplanted tumor for- mation numbers.

Article Snippet: The primary antibodies were incubated at 4 °C overnight: anti-AKT (dilution 1:1000), anti-AKT1 (1:1000), p-AKT (Ser473) (1:1000), p-AKT (Thr308) (1:1000) and Vimentin (1:500) were purchased from CST, βcatenin (1:2000), c-Myc (1:1000) from Proteintech, ALDH1A1 (1:1000, AL: ALDH1A1; L: low expression; H: High expression; HEs: Hyperplastic esophagus tissues; ESCCs: Esophageal squamous cell carcinoma tissues.

Techniques: Expressing, In Vivo, Injection

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: ALDH1A1 maintains the cancer stem-like cells properties of esophageal squamous cell carcinoma by activating the AKT signal pathway and interacting with β-catenin.

doi: 10.1016/j.biopha.2020.109940

Figure Lengend Snippet: Fig. 4. ALDH1A1 expression regulated AKT and β-catenin expressions. (A) RT-qPCR assay detected mRNA expression levels of ALDH1A1, AKT1, β-catenin, Slug, c-Myc, and Vimentin in KYSE-150 and TE-1 cells. ** P < 0.01; *** P < 0.001; ns, not significant. (B, C) Western blot assay showed the ALDH1A1, p-AKT, total-AKT1, β-catenin, c-Myc, and β-actin protein expressions after NCT501 treatment or ALDH1A1 overexpression in ESCC cells, *: GAPDH. D, Western blot assay showed the ALDH1A1, AKT1, β-catenin, and β-actin expression levels after MK-2206 or NCT501 treatment. (E) Western blot gray scale for the relative protein levels of Fig. 4E. ***: P < 0.001.

Article Snippet: The primary antibodies were incubated at 4 °C overnight: anti-AKT (dilution 1:1000), anti-AKT1 (1:1000), p-AKT (Ser473) (1:1000), p-AKT (Thr308) (1:1000) and Vimentin (1:500) were purchased from CST, βcatenin (1:2000), c-Myc (1:1000) from Proteintech, ALDH1A1 (1:1000, AL: ALDH1A1; L: low expression; H: High expression; HEs: Hyperplastic esophagus tissues; ESCCs: Esophageal squamous cell carcinoma tissues.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: ALDH1A1 maintains the cancer stem-like cells properties of esophageal squamous cell carcinoma by activating the AKT signal pathway and interacting with β-catenin.

doi: 10.1016/j.biopha.2020.109940

Figure Lengend Snippet: Fig. 5. ALDH1A1 interacted with β-catenin. Co-localization of ALDH1A1 and β-catenin in (A) adherent cells (Scale bar =20 μm) and (B) stem cell spheres (Scale bar =50 μm) of KYSE-510. Images were collected by a confocal microscope. (C) Co-IP, Western blot assay to detect the interaction of ALDH1A1 and β-catenin. (D) Western blot to detect β-catenin protein levels after cycloheximide (CHX) treatment. (E) Western blot to detect ALDH1A1and β-catenin protein expression in cytoplasm and nuclear of adherent KYSE-510 cells. (F) TOP/FOP flash activity assay revealed that ALDH1A1 promoted wnt/β-catenin activity in KYSE-293 T cells.

Article Snippet: The primary antibodies were incubated at 4 °C overnight: anti-AKT (dilution 1:1000), anti-AKT1 (1:1000), p-AKT (Ser473) (1:1000), p-AKT (Thr308) (1:1000) and Vimentin (1:500) were purchased from CST, βcatenin (1:2000), c-Myc (1:1000) from Proteintech, ALDH1A1 (1:1000, AL: ALDH1A1; L: low expression; H: High expression; HEs: Hyperplastic esophagus tissues; ESCCs: Esophageal squamous cell carcinoma tissues.

Techniques: Microscopy, Co-Immunoprecipitation Assay, Western Blot, Expressing, Activity Assay

Journal: Cell reports

Article Title: Phase-separated nuclear bodies of nucleoporin fusions promote condensation of MLL1/CRM1 and rearrangement of 3D genome structure.

doi: 10.1016/j.celrep.2023.112884

Figure Lengend Snippet: Figure 3. FG repeat and homeodomain of NUP98::HOXA9 are important for its accumulation on HOX clusters and gene activation (A and B) Subcellular localization of NUP98:HOXA9 and Hoxa or Outside (control: 200 kb distant region from cdkn2a) gene loci in NUP98::HOXA9 ESCs. (A) ImmunoFISH was performed to detect NUP98::HOXA9 nuclear bodies and respective gene loci. The open arrow indicates a co-localized locus. Scale bars, 5 mm. (B) The minimum distance between each gene locus (as revealed by FISH) and the closest NUP98::HOXA9 nuclear bodies were analyzed using ImageJ (n = 541 for Hoxa, n = 274 for Outside). (C) Schematic representation of NUP98::HOXA9, FG mutant (AG), homeodomain mutant (N51S), or GLFG mutant (GAFG). Yellow bars, asterisk, or blue bars indicate the mutated amino acid residues in the respective construct.

Article Snippet: As for CRM1, we previously demonstrated that ChIP-seq peaks of CRM1were significantly enhanced in FLAG-NUP98::HOXA9-expressing cells compared with parental ESCs using a polyclonal anti-CRM1 antibody (Bethyl Labs).33 Here, we noticed that ChIP-seq using a monoclonal anti-CRM1 antibody (CST) showed marked differences in CRM1 ChIP signals between parental ESCs and FLAG-NUP98::HOXA9-expressing cells (Figures 4B–4E).

Techniques: Activation Assay, Control, Mutagenesis, Construct

Journal: PloS one

Article Title: Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

doi: 10.1371/journal.pone.0090454

Figure Lengend Snippet: Figure 3. Proliferation of GSC-like cells through culture with GDNF, bFGF and LIF (A) Total numbers and proliferation activity of embryoid body (EB) cells (from CHA-hES15 and H1 cell lines) in cultured groups with EB medium+BMP4/RA, EB medium only or GC proliferation medium for 1 and 4 weeks. (B) Flow cytometric analysis of GFR-a1 expression in ESC-derived GSC-like cells (from CHA-hES15 and H1 cell lines) cultured in EB medium+BMP4/RA, EB medium only or GC proliferation medium for 1 and 4 weeks. (C) Immunocytochemical staining of GFR-a1 expression in ESC- derived GSC-like cells (from CHA-hES15 and H1 cell lines) cultured in EB medium+BMP4/RA, EB medium only or GC proliferation medium for 1 and 4 weeks. Note: a vs b vs c, p,0.05, Scale bars are 50 mm. doi:10.1371/journal.pone.0090454.g003

Article Snippet: Immunocytochemical analysis To investigate the localization and expression of ESC and GSC markers in cultured colonies or MACS-sorted cells, we performed immunocytochemistry using antibodies [OCT4 (Santa Cruz Biotechnology, Santa Cruz, CA), SSEA-4 (Chemicon), TRA-160 (Chemicon) and SSEA-3 (Chemicon) were used for ESC; integrin a6 (Santa Cruz Biotechnology) and integrin b1 (Santa Cruz Biotechnology) were used for ESC or GSC; VASA (R&D Systems) and GFR-a1 (Chemicon) were used for GSC or GSC-like cells; intra-acrosomal protein (IAP, Abcam) was used for spermatid [25].

Techniques: Activity Assay, Cell Culture, Expressing, Derivative Assay, Staining

Journal: PloS one

Article Title: Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

doi: 10.1371/journal.pone.0090454

Figure Lengend Snippet: Figure 5. In vitro culture of GSC-like cells using a 3D-co-culture system and the potential for differentiation into germ cell lineages. (A) Expression of stage-specific genes (integrin a6, c-Kit, TH2B and TP-1) during in vitro differentiation of human ESC-derived GSC-like cells using RT- PCR. Integrin a6 (undifferentiated spermatogonia), c-Kit and TH2B (differentiating spermatogonia and spermatocytes), TP-1 (meiotic spermatocytes and spermatids) were used as stage-specific germ cell marker genes. 18S ribosomal RNA was used as a control gene. Line 1–3: ESC-derived GSC-like cells (0, 1, and 2-week culture in the 3D-co-culture system), N: no reverse transcriptase. (B) DNA content of in vitro differentiated ESC-derived GSC-like cells. a; DNA content of cells from obstructive azoospermia (OA, normal spermatogenesis sample) b; Undifferentiated human embryonic stem cells (CHA-hES15). N1 represents haploid germ cells and N2 represents diploid cells. (C) Localization of intra acrosomal protein (IAP) in human testes (upper panel) and in vitro differentiated GSC-like cells from hESCs (middle and lower figure). IAP was stained in spermatid acrosomes. The yellow arrows indicate fluorescence signals. Note: **; significantly different (p,0.05). Scale bars: 50 mm. doi:10.1371/journal.pone.0090454.g005

Article Snippet: Immunocytochemical analysis To investigate the localization and expression of ESC and GSC markers in cultured colonies or MACS-sorted cells, we performed immunocytochemistry using antibodies [OCT4 (Santa Cruz Biotechnology, Santa Cruz, CA), SSEA-4 (Chemicon), TRA-160 (Chemicon) and SSEA-3 (Chemicon) were used for ESC; integrin a6 (Santa Cruz Biotechnology) and integrin b1 (Santa Cruz Biotechnology) were used for ESC or GSC; VASA (R&D Systems) and GFR-a1 (Chemicon) were used for GSC or GSC-like cells; intra-acrosomal protein (IAP, Abcam) was used for spermatid [25].

Techniques: In Vitro, Co-Culture Assay, Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Control, Reverse Transcription, Staining, Fluorescence

Journal: PloS one

Article Title: Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

doi: 10.1371/journal.pone.0090454

Figure Lengend Snippet: Figure 6. Comparative analyses between ESC-derived GSC-like cells and testis-derived spermatogonia using known spematogonial marker genes (A) The expression profiles of the 118 genes in the 2 types of cells are presented as a Venn diagram and table. a; A total of 52 genes were more strongly expressed in testis-derived SSCs than in ESC-derived GSC-like cells. b; A total of 48 genes were similarly expressed in the 2 groups. c; A total of 18 genes were more strongly expressed in ESC-derived GSC-like cells than in testis-derived spermatogonia. (B) The validation of randomly selected spematogonial marker genes using real-time RT-PCR. Note: In vitro cultured ESC-derived GSC-like cells (at 2nd passage); In vitro cultured testis-derived spermatogonia (at 2nd passage). doi:10.1371/journal.pone.0090454.g006

Article Snippet: Immunocytochemical analysis To investigate the localization and expression of ESC and GSC markers in cultured colonies or MACS-sorted cells, we performed immunocytochemistry using antibodies [OCT4 (Santa Cruz Biotechnology, Santa Cruz, CA), SSEA-4 (Chemicon), TRA-160 (Chemicon) and SSEA-3 (Chemicon) were used for ESC; integrin a6 (Santa Cruz Biotechnology) and integrin b1 (Santa Cruz Biotechnology) were used for ESC or GSC; VASA (R&D Systems) and GFR-a1 (Chemicon) were used for GSC or GSC-like cells; intra-acrosomal protein (IAP, Abcam) was used for spermatid [25].

Techniques: Derivative Assay, Marker, Expressing, Biomarker Discovery, Quantitative RT-PCR, In Vitro, Cell Culture

Journal: Frontiers in immunology

Article Title: Hypoimmunogenic CD19 CAR-NK cells derived from embryonic stem cells suppress the progression of human B-cell malignancies in xenograft animals.

doi: 10.3389/fimmu.2024.1504459

Figure Lengend Snippet: FIGURE 1 Generation of CD19 CAR-UESC and induction of CD19 CAR-UiNK. (A) Schematic diagram of CD19 CAR-UiNK generation and functional assay. (B) Targeting strategy for the knockout of B2M gene in human ESCs. The exons of the B2M gene are shown as blue boxes. Two gRNA sequences, gRNA1 and gRNA2, are marked in red. (C) Schematic representation of the transposon vector encoding the CD19 CAR. (D) Schematic representation of the transposon vector encoding the HLA-E single chain trimer. (E) Representative flow cytometry plots showing the expression of HLA-ABC, HLA-E, and CD19 CAR in ESC and CD19 CAR-UESC. (F) Schematic diagram of CD19 CAR-UiNK cell induction. (G) Representative flow cytometry plots of CD19 CAR-UiNK cells (CD45+CD3–CD56+CD16+/–HLA-ABC–HLA-E+CD19 CAR+) on Day 27.

Article Snippet: After editing, the ESCs were incubated with FITC anti-human HLA-ABC antibody (clone: W6/32, Biolegend, 311404) on ice for 15-30 min. HLA-ABC negative ESCs were sorted by Sony MA900 Sorter.

Techniques: Functional Assay, Knock-Out, Plasmid Preparation, Cytometry, Expressing

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: PDIA3P1 serves as a promoter of glycolysis in esophageal squamous cell carcinoma. A) KEGG metabolism analysis of the differential genes of shPDIA3P1 versus shNC by RNA‐seq. B,C) 2‐NBDG uptake was measured by flow cytometry to monitor glucose uptake in cells with transfection PDIA3P1‐siRNA (B) or PDIA3P1 expression plasmid (C) (MFI: Mean fluorescence intensity). D,E) Lactate production was measured in silenced PDIA3P1 (D) or overexpressed PDIA3P1 cells (E). F,G) Detection of glucose uptake in knocking down PDIA3P1 cells (F) or overexpressing PDIA3P1 cells (G). H,I) Seahorse metabolic analysis of ECAR, glycoPER (glycolytic proton efflux rate), basal glycolysis and compensatory glycolysis in PDIA3P1 knockdown cells (H) or PDIA3P1 overexpression cells (I) (Rot/AA: Rotenone/Antimycin A; 2‐DG: 2‐Deoxy‐D‐glucose). J,K) Relative level of acetyl‐CoA was measured in silenced PDIA3P1 cells (J) or overexpressed PDIA3P1 cells (K). L,M) Detection of relative α‐ketoglutarate levels in knocking down PDIA3P1 cells (L) or overexpressing PDIA3P1 cells (M). These data represent the mean ± S.D. of triplicates. ns: no significance; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: RNA Sequencing, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Fluorescence, Knockdown, Over Expression

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: Glycolysis mediates PDIA3P1 regulation of esophageal squamous cell carcinoma progression. A) Schematic diagram of glycolysis and inhibition methods target. B–G) Stable silencing of PDIA3P1 in TE‐1 and Eca‐109 cells was treated with exogenous lactate (15m m ) for 24h. B) The proliferative abilities were investigated via CCK‐8 assays. C) ESCC cell growth was analyzed using colony formation assay. The upper panel is for statistical analysis. D) EdU assays were performed to assess the proliferative ability of ESCC cells. The upper panel is for statistical analysis. Scale bar: 100 µm. E) Annexin V‐APC/7‐AAD staining for detecting apoptosis in TE‐1 and Eca‐109 cells by flow cytometry. The right panel is for statistical analysis. F) Transwell assays were used to detect changes in the cell migration and invasion. The upper panel is for statistical analysis. Transwell Scale bar: 10 µm. G) Western blot shows expression levels of E‐Cadherin, N‐Cadherin, Vimentin, and Snail. These data represent the mean ± S.D. of triplicates. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Inhibition, CCK-8 Assay, Colony Assay, Staining, Flow Cytometry, Migration, Western Blot, Expressing

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: PDIA3P1 upregulates GLUT1 and HK2 to promote the glycolysis in ESCC cells. A,B) Western blot analysis of GLUT1, HK2, PFKFB3, PKM2, and LDHA after PDIA3P1 stable silencing in TE‐1 and Eca‐109 cells (A) or PDIA3P1 stable overexpressing in KYSE‐30 and KYSE‐150 cells (B). C,E,G,I) In Eca‐109 cells with stable knockdown of PDIA3P1, overexpression plasmids of GLUT1 and HK2 were transfected separately. D,F,H,J) In KYSE‐150 cells stably overexpressing PDIA3P1, GLUT1 siRNA and HK2 siRNA were transfected separately. C,D) Relative 2‐NBDG uptake detected by flow cytometry (MFI: Mean fluorescence intensity). E,F) Relative lactate production was measured using lactate assay kit. G,H) Detection of relative glucose uptake in ESCC. I,J) Cells were subjected to a Seahorse metabolic analyzer to determine the glycolytic rate including ECAR, glycoPER, basal glycolytic rate, and compensatory glycolytic rate. These data represent the mean ± S.D. of triplicates. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Western Blot, Knockdown, Over Expression, Transfection, Stable Transfection, Flow Cytometry, Fluorescence, Lactate Assay

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: PDIA3P1 acted as a sponge of miR‐152‐3p to regulate GLUT1 expression. A,B) qRT‐PCR analyses of GLUT1 mRNA levels in stably PDIA3P1‐KD cells (A) or stably PDIA3P1‐OE cells (B). C) RIP assay for AGO2 was conducted to detect the levels of endogenous PDIA3P1 in the IP pellet of AGO2. D) The miRNAs that sponged by PDIA3P1 was predicted by miRNet and ENCORI and miRNAs that targeting GLUT1 by miRNet, TargetScan, miRDB, and ENCORI, the intersection predicted five miRNAs. E,F) AGO2‐RIP assays showed the enrichment of the predicted five miRNAs in stably PDIA3P1‐KD cells (E) or stably PDIA3P1‐OE cells (F). G) Putative binding sequence between PDIA3P1 and miR‐152‐3p. H) TE‐1 and Eca‐109 cells co‐transfected with wild‐type or mutant lncRNA PDIA3P1 and miR‐152‐3p mimics or control were detected by dual luciferase reporter assay. I) Luciferase reporters containing WT or MUT PDIA3P1 transcript were co‐transfected with miR‐152‐3p inhibitor or miR‐control in KYSE‐30 and KYSE‐150 cells. J) FISH results showing the colocalization of PDIA3P1 and miR‐152‐3p in cytoplasm of Eca‐109 cells. K) Enrichment of PDIA3P1 pulled down by biotin‐miR‐152‐3p or biotin‐miR‐control. L) Putative binding sequence of miR‐152‐3p in the 3′‐UTR of GLUT1. M) ESCC cells co‐transfected with wild‐type or mutant 3′‐UTR of GLUT1 and miR‐135b‐3p mimics or control were detected by dual luciferase reporter assay. N) Luciferase reporters containing WT or MUT 3′‐UTR of GLUT1 were co‐transfected with miR‐152‐3p inhibitor or miR‐control in ESCC cells. O) Transfection of miR‐152‐3p inhibitor into stably PDIA3P1‐KD cells, the expression levels of GLUT1 were detected by qRT‐PCR. P) Transfection of miR‐152‐3p mimics into stably PDIA3P1‐OE cells, the expression levels of GLUT1 were detected by qRT‐PCR. Q, R) Transfection of miR‐152‐3p inhibitor into stably PDIA3P1‐KD cells (Q) or miR‐152‐3p mimics into stably PDIA3P1‐OE cells (R), the expression levels of GLUT1 were detected by Western blot. These data represent the mean ± S.D. of triplicates. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Expressing, Quantitative RT-PCR, Stable Transfection, Binding Assay, Sequencing, Transfection, Mutagenesis, Control, Luciferase, Reporter Assay, Western Blot

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: PDIA3P1 increases H4K8la level by promoted glycolysis in ESCC cells. A) Western blot shows the levels of Pan Kla in normal cell line HEEC and five ESCC cell lines (KYSE‐30, KYSE‐150, KYSE‐520, TE‐1, and Eca‐109). B,C) Western blot analysis of Pan‐ Kla after PDIA3P1 stable silencing in TE‐1 and Eca‐109 cells (B) or PDIA3P1 stable overexpressing in KYSE‐30 and KYSE‐150 cells (C). D,E) Representative images of IF staining revealing the effect of PDIA3P1 knockdown in Eca‐109 (D) or PDIA3P1 overexpressed in KYSE‐150 (E) on the expression of Pan‐ Kla. F, G) Western blot analysis of site‐specific histone lactylation in PDIA3P1‐KD cells (F) or PDIA3P1‐OE cells (G). H, I) Representative images of IF staining revealing the effect of PDIA3P1 knockdown in Eca‐109 (H) or PDIA3P1 overexpressed in KYSE‐150 (I) on the expression of H4K8la. J) PDIA3P1‐KD cells were treated with lactate for 24 h, Western blot measured the level of H4K8 lactylation. K) H4K18la levels were detected in PDIA3P1‐OE cells cultured in 2‐DG or oxamate for 24 h by western blot.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Western Blot, Staining, Knockdown, Expressing, Cell Culture

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: BMP7 is a target of H4K8 lactylation in ESCC cells. A) The binding density of H4K8la was visualized by deepTools: the heatmap illustrates the CUT&Tag tag counts on the various H4K8la enrichment peaks in shNC and shPDIA3P1 cells. B) Distribution of H4K8la sites relative to translation start site (TSS). C Genome‐wide distribution of downregulated H4K8la‐binding peaks in shPDIA3P cells. D) KEGG analysis of the decreased H4K8la binding peaks at candidate target genes. E) Transcriptome sequencing was performed in control and shPDIA3P1 cells. F) Venn Diagram of CUT&Tag, RNA‐seq, GEO, and pubmed database to identify the potential downstream targets of H4K8la. G) Integrative Genomics Viewer tracks of CUT&Tag showing enriched H4K8la in the promotors of BMP7. The red rectangles indicate the peak regions of H4K8la on target‐gene promoters. H‐K) BMP7 mRNA and protein levels were measured in PDIA3P1‐KD cells (H, J) or PDIA3P1‐OE cells (I, K). L) Western blotting analysis of BMP7 expression in PDIA3P1‐KD cells cultured in lactate for 24 h. M) Western blot analysis of BMP7 expression in PDIA3P1‐OE cells cultured in 2‐DG or oxamate for 24 h. N, O) Using antibodies against H4K8la, CUT&Tag‐qPCR analysis for binding status at the BMP7 promotor of PDIA3P1‐KD cells (N) or PDIA3P1‐OE cells (O). These data represent the mean ± S.D. of triplicates. ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Binding Assay, Genome Wide, Sequencing, Control, RNA Sequencing, Western Blot, Expressing, Cell Culture

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: PDIA3P1 promotes tumorigenesis of esophageal squamous cell carcinoma through BMP7 both in vitro and in vivo. A) Western blot detection of BMP7 expression to reflect the effect of transfection of BMP7 by lentivirus. B‐G) Using lentivirus to construct BMP7 stable overexpression cell lines in TE‐1 and Eca‐109 cells with stably PDIA3P1‐KD. B) Proliferation of cells was analyzed using CCK8 assay. C) Tumor growth of TE‐1 and Eca‐109 cells was evaluated by colony formation assay. D) The proliferative abilities of ESCC cells were investigated using EdU assays. EdU scale bar: 100 µm. E) The flow cytometry showing apoptosis of cells by Annexin V‐APC and 7‐AAD staining. F) The migration and invasion ability was assessed by transwell assays. Transwell scale bar: 10 µm. G) Western blot shows expression levels of E‐Cadherin, N‐Cadherin, Vimentin, Snail, and BMP7. H‐K) Eca‐109 cells transfected with control, shPDIA3P1 or shPDIA3P1 and BMP7 were injected subcutaneously to establish a tumorigenesis model in nude mice ( n = 6). H) Photograph and comparison of tumor sizes in the indicated groups. I) The parameters of subcutaneous tumors were measured and recorded every 5 days, and calculated the volume of the tumor according to the formula below: tumor volume = 0.5 × length × width × width (mm3). J) Tumor weights in the indicated groups. K) Ki‐67 and BMP7 expression of representative IHC of nude mice tumor tissues. Scale bar, 20 µm. IF staining of TUNEL in subcutaneous tumor tissue sections. Scale bar: 50 µm. These data represent the mean ± S.D. of triplicates. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Transfection, Construct, Over Expression, Stable Transfection, CCK-8 Assay, Colony Assay, Flow Cytometry, Staining, Migration, Control, Injection, Comparison, TUNEL Assay

Journal: Advanced Science

Article Title: WTAP Mediated m6A Modification Stabilizes PDIA3P1 and Promotes Tumor Progression Driven by Histone Lactylation in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202506529

Figure Lengend Snippet: Increased stability of PDIA3P1 is via IGF2BP1 recognition of WTAP‐mediated m6A modification. A) The enriched and specific m6A peak distribution of PDIA3P1 predicted by SRAMP. B) MeRIP‐qPCR analysis of m6A modification level of PDIA3P1 in normal cell line HEEC and five ESCC cell lines (KYSE‐30, KYSE‐150, KYSE‐520, TE‐1, and Eca‐109). C) Correlation analysis showing a positive correlation between WTAP and PDIA3P1 expression. D) qRT‐PCR analysis of PDIA3P1 expression in TE‐1 and Eca‐109 cells with WTAP knockdown. E) Relative luciferase activity in TE‐1 and Eca‐109 cells co‐transfected with luciferase reporter pmirGLO‐PDIA3P1 and WTAP siRNA. F) WTAP silencing cells treated with actinomycin D (10 µg mL −1 ) for the various time points; the level of PDIA3P1 was examined by qRT‐PCR. G) The m6A modification level of PDIA3P1 was examined in WTAP‐silencing cells by MeRIP‐qPCR. H) PDIA3P1 pull‐down followed by Western blot validated its interaction with WTAP. I) RIP assay was performed using the WTAP antibody in KYSE‐150 and Eca‐109 cells. J) Relative luciferase activity of the wild‐type and its mutants pmirGLO‐PDIA3P1 reporter vectors when transfected with WTAP siRNA in TE‐1 and Eca‐109 cells (WT: wild‐type; A–G mut: adenine residues substituted by guanine; A–T mut: adenine residues substituted by thymine; A–Del mut: adenine residues deleted). K) Relative luciferase activity of the wild‐type and its mutants pmirGLO‐PDIA3P1 reporter vectors during IGF2BP1 silencing in ESCC cells. L) PDIA3P1 stability in control and IGF2BP1‐silenced cells, the level of PDIA3P1 was examined by qRT‐PCR. M) PDIA3P1 pull‐down followed by Western blot validated its interaction with IGF2BP1. N) RIP assay was performed using the IGF2BP1 antibody. O) FISH and IF double staining showing the co‐localization of PDIA3P1 and IGF2BP1 in the cytoplasm. P) qRT‐PCR analysis of PDIA3P1 expression in KYSE‐30 and KYSE‐150 cells co‐transfected with WTAP overexpression vector and the IGF2BP1 siRNA. Q) Relative luciferase activity in KYSE‐30 and KYSE‐150 cells co‐transfected with WTAP overexpression vector and the IGF2BP1 siRNA. R) PDIA3P1 stability in ESCC cells co‐transfected with WTAP overexpression vector and the IGF2BP2 siRNA, the level of PDIA3P1 was examined by qRT‐PCR. S) Transfection or co‐transfection of OCT4 cDNA or WTAP cDNA separately, and expression of PDIA3P1 was detected by qRT‐PCR. These data represent the mean ± S.D. of triplicates. ns: no significance; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Stably PDIA3P1‐knockdown (KD) ESCC cells were selected using 500μg mL −1 G‐418 (HY‐K1056, MCE).

Techniques: Modification, Expressing, Quantitative RT-PCR, Knockdown, Luciferase, Activity Assay, Transfection, Western Blot, Control, Double Staining, Over Expression, Plasmid Preparation, Cotransfection

Journal: Clinics (Sao Paulo, Brazil)

Article Title: The role of TMEM26 in disrupting tight junctions and activating NF-κB signaling to promote epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

doi: 10.1016/j.clinsp.2023.100276

Figure Lengend Snippet: Fig. 3. TMEM26 depletion in ESCC cells suppressed EMT-related alterations. (A) Transfection of siRNA targeting the TMEM26 gene (three independent targets) effi- ciently downregulated TMEM26 expression in TMEM26-high KYSE270 and TE8 cells, as detected in western blotting. (B) Transwell assay revealed a reduced number of invaded cells with TMEM26 RNAi, compared with control RNAi, in TMEM26-high KYSE270, and TE8 cells. Left: representative images. Right: quantification and sta- tistical result. Scale bar = 100 µm. (C) Wound healing assay for TMEM26 RNAi KYSE270 and TE8 cells showed fewer cells migrating into the wound region, compared with control RNAi cells. Left: representative images. Right: quantification and statistical result. Scale bar = 100 µm. (D) Western blotting to detect mesenchymal and epithelial marker expressions showed downregulated twist, snail, N-cadherin, and vimentin but upregulated E-cadherin in TMEM26 RNAi KYSE270 and TE8 cells. Data are presented as means ± S.D., n = 3, ***p < 0.001.

Article Snippet: Cell lines, reagents, and antibodies ESCC cell lines (KYSE150, KYSE270, KYSE450, TE4, and TE8) and a normal esophageal epithelial cell line (HET-1A) were obtained from ATCC (MA, USA).

Techniques: Transfection, Expressing, Western Blot, Transwell Assay, Control, Wound Healing Assay, Marker

Journal: Clinics (Sao Paulo, Brazil)

Article Title: The role of TMEM26 in disrupting tight junctions and activating NF-κB signaling to promote epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

doi: 10.1016/j.clinsp.2023.100276

Figure Lengend Snippet: Fig. 2. TMEM26 expression and cellular localization in various ESCC cell lines. (A) The abundance of TMEM26 in the normal esophageal epithelial cell line (HET-1A) and a collection of ESCC cell lines (KYSE150, KYSE270, KYSE450, TE4, and TE8) were examined by western blotting. (B) Immunofluorescent staining of TMEM26 with DAP in KYSE270 and TE8 cells verified the plasma membrane localization of TMEM26. Scale bar = 10 µm.

Article Snippet: Cell lines, reagents, and antibodies ESCC cell lines (KYSE150, KYSE270, KYSE450, TE4, and TE8) and a normal esophageal epithelial cell line (HET-1A) were obtained from ATCC (MA, USA).

Techniques: Expressing, Western Blot, Staining, Clinical Proteomics, Membrane

Journal: Clinics (Sao Paulo, Brazil)

Article Title: The role of TMEM26 in disrupting tight junctions and activating NF-κB signaling to promote epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

doi: 10.1016/j.clinsp.2023.100276

Figure Lengend Snippet: Fig. 4. TMEM26 overexpression in ESCC cells promoted EMT-related alterations. (A) Transfection of TMEM26-expressing plasmid by lentiviral vectors efficiently over- regulated TMEM26 in TMEM26-low KYSE150 and KYSE450 cells, as shown by western blotting. (B) Transwell assay showed a higher number of invaded cells with TMEM26 overexpression than with control transfection in TMEM26-low KYSE150 and KYSE450 cells. Left: representative images. Right: quantification and statistical result. Scale bar = 100 µm. (C) The wound healing assay showed that the number of cells migrating into the wound region in TMEM26-overexpressing KYSE150 and KYSE450 cells was higher than that in control transfection cells. Left: representative images. Right: quantification and statistical result. (D) Western blotting detection of mesenchymal and epithelial marker expressions showed upregulated twist, snail, N-cadherin, and vimentin but downregulated E-cadherin in TMEM26-overexpress- ing KYSE150 and KYSE450 cells than that in control transfection cells. Data are presented as means ± S.D., n = 3, ***p < 0.001.

Article Snippet: Cell lines, reagents, and antibodies ESCC cell lines (KYSE150, KYSE270, KYSE450, TE4, and TE8) and a normal esophageal epithelial cell line (HET-1A) were obtained from ATCC (MA, USA).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Transwell Assay, Control, Wound Healing Assay, Marker

Journal: Clinics (Sao Paulo, Brazil)

Article Title: The role of TMEM26 in disrupting tight junctions and activating NF-κB signaling to promote epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

doi: 10.1016/j.clinsp.2023.100276

Figure Lengend Snippet: Fig. 5. TMEM26 did not affect ESCC cell growth. (A) In TE8 (left) and KYSE270 (right) cells, siRNA knockdown of TMEM26 (three independent targets) did not show observable differences in the growth curve compared with siRNA control cells, as determined by MTT assay. (B) In KYSE150 (left) and KYSE450 (right) cells, TMEM26 overexpression due to lentiviral transfection did not show observable differences in the growth curve compared with control cells, as determined by MTT assay. Data are presented as means ± S.D., n = 6.

Article Snippet: Cell lines, reagents, and antibodies ESCC cell lines (KYSE150, KYSE270, KYSE450, TE4, and TE8) and a normal esophageal epithelial cell line (HET-1A) were obtained from ATCC (MA, USA).

Techniques: Knockdown, Control, MTT Assay, Over Expression, Transfection

Journal: Clinics (Sao Paulo, Brazil)

Article Title: The role of TMEM26 in disrupting tight junctions and activating NF-κB signaling to promote epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

doi: 10.1016/j.clinsp.2023.100276

Figure Lengend Snippet: Fig. 7. Detection for NF-κB signaling activation by TMEM26 and the determining effects of NF-κB signaling on EMT in ESCC cells. (A) In TMEM26-high TE8 (left) and KYSE270 (right) cells, siRNA knockdown of TMEM26 (three independent targets) did not show observable differences for the total number of p65 and IκBα; however, the p65 and IκBα phosphorylation was dra- matically declined, as detected in western blotting. (B) TMEM26 overexpression in TMEM26-low KYSE150 and KYSE450 cells resulted in elevated p65 and IκBα phosphorylation, whereas the total amount of p65 and IκBα remained comparable, as detected in western blotting. (C) In KYSE150 and KYSE450 cells, TMEM26 overexpression enhanced the expression of twist; how- ever, the treatment with PS1145, an NF-κB inhibitor, blocked this effect, as detected in western blotting. (D) Wound healing assay for TMEM26-overexpressing KYSE150 and KYSE450 cells showed a higher number of cells migrating into the wound region, which was blocked by PS1145. Left: representative images. Right: quantification and statistical result. Scale bar = 100 µm. (E) Transwell assay for KYSE150 and KYSE450 cells showed a higher number of invading cells after overexpressing TMEM26, which was blocked by PS1145. Right: quantification and statisti- cal result. Scale bar = 100 µm. Data are presented as means ± S.D., n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001 versus the vector group, ###p < 0.001 vs. the TMEM26 OE group.

Article Snippet: Cell lines, reagents, and antibodies ESCC cell lines (KYSE150, KYSE270, KYSE450, TE4, and TE8) and a normal esophageal epithelial cell line (HET-1A) were obtained from ATCC (MA, USA).

Techniques: Activation Assay, Knockdown, Phospho-proteomics, Western Blot, Over Expression, Expressing, Wound Healing Assay, Transwell Assay, Plasmid Preparation

Journal: Clinics (Sao Paulo, Brazil)

Article Title: The role of TMEM26 in disrupting tight junctions and activating NF-κB signaling to promote epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

doi: 10.1016/j.clinsp.2023.100276

Figure Lengend Snippet: Fig. 8. Regulation of plasma membrane distribution of TJ proteins by TMEM26 in TE8 and KYSE150 ESCC cells. (A and B) Immunofluorescent staining for claudin-1o Occludin, and ZO-1 with DAPI in TE8 cells showed their plasma membrane localization. Compared with the siRNA control, the TE8 cells with siRNA knockdown of TMEM26 (three independent targets) elevated the distribution of claudin-1, occludin, and ZO-1 at the plasma membrane. Quantification of plasma membrane signal intensity is shown in (B). (C and D) Immunofluorescent staining for claudin-1, occludin, and ZO-1 with DAPI in KYSE150 cells showed their plasma membrane localiza- tion. Compared with control transfection, KYSE150 cells with overexpressed TMEM26 showed a reduced distribution of claudin-1, occludin, and ZO-1 at the plasma membrane. Quantification of plasma membrane signal intensity is shown in (D). Scale bar = 10 µm. Data are presented as means ± S.D., n = 3, **p < 0.01, and ***p < 0.001.

Article Snippet: Cell lines, reagents, and antibodies ESCC cell lines (KYSE150, KYSE270, KYSE450, TE4, and TE8) and a normal esophageal epithelial cell line (HET-1A) were obtained from ATCC (MA, USA).

Techniques: Clinical Proteomics, Membrane, Staining, Control, Knockdown, Transfection

Journal: PLoS ONE

Article Title: Variations in the MHC Region Confer Risk to Esophageal Squamous Cell Carcinoma on the Subjects from High-Incidence Area in Northern China

doi: 10.1371/journal.pone.0090438

Figure Lengend Snippet: C (10X) was the negative control of A (omission of the first antibody application). A, B and C were the serial tissue sections from the same ESCC patient. E and F were also from the same patient.

Article Snippet: Sixty-seven ESCC cases with 60 matched normal tissues (7 pieces of tissue were off-chip during the histological dehydration process) for TRIM27 immunostaining and 49 ESCC cases with 41 matched normal tissues (8 pieces of tissue were off-chip during the histological dehydration process) for HLA-DQA1 immunostaining (ProteinTech Group, Inc., Chicago, USA).

Techniques: Negative Control

Journal: PLoS ONE

Article Title: Variations in the MHC Region Confer Risk to Esophageal Squamous Cell Carcinoma on the Subjects from High-Incidence Area in Northern China

doi: 10.1371/journal.pone.0090438

Figure Lengend Snippet: Relationship of TRIM27 protein immunostaining and clinical characteristics of ESCC.

Article Snippet: Sixty-seven ESCC cases with 60 matched normal tissues (7 pieces of tissue were off-chip during the histological dehydration process) for TRIM27 immunostaining and 49 ESCC cases with 41 matched normal tissues (8 pieces of tissue were off-chip during the histological dehydration process) for HLA-DQA1 immunostaining (ProteinTech Group, Inc., Chicago, USA).

Techniques: Immunostaining

Journal: PLoS ONE

Article Title: Variations in the MHC Region Confer Risk to Esophageal Squamous Cell Carcinoma on the Subjects from High-Incidence Area in Northern China

doi: 10.1371/journal.pone.0090438

Figure Lengend Snippet: Relationship of HLA-DQA1 protein immunostaining and clinical characteristics of ESCC.

Article Snippet: Sixty-seven ESCC cases with 60 matched normal tissues (7 pieces of tissue were off-chip during the histological dehydration process) for TRIM27 immunostaining and 49 ESCC cases with 41 matched normal tissues (8 pieces of tissue were off-chip during the histological dehydration process) for HLA-DQA1 immunostaining (ProteinTech Group, Inc., Chicago, USA).

Techniques: Immunostaining