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 Death & Disease

Article Title: Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration

doi: 10.1038/s41419-022-05199-5

Figure Lengend Snippet: The expression level of PTPN13 in shCont-iRPE and shPtpn13-iRPE cells were detected by A Western blotting and B quantitative analysis ( n = 3, P value measured by Student’s unpaired t test). C Representative images of shCont-iRPE and shPtpn13-iRPE cells. RPE-specific and EMT-associated markers in shCont-iRPE cells and shPtpn13-iRPE cells stimulated with TGF-β1 or TGF-β2 were detected by D immunostaining, E Western blotting, and F quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). Phosphorylation of SMAD2/3, p38, and ERK1/2 in shCont-iRPE and shPtpn13-iRPE cells were analyzed by G immunostaining, H Western blotting, and I quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). Scale bar = 50 μm. Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, shPtpn13-TGF-β1 compared with shCont-TGF-β1; # P < 0.01, ## P < 0.01, ### P < 0.001, shPtpn13-TGF-β2 compared with shCont-TGF-β2.

Article Snippet: After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against RPE65 (1:1000, Novus Biologicals, Centennial, CO, USA); against TYRP1 (1:1000), MERTK (1:1000), FN1 (1:1000), α-SMA (1:2000), pSMAD2 (1:500), SMAD2 (1:1000), pSMAD3 (1:1000), SMAD3 (1:1000) TGF-β receptor1 (TGF-βR1) (1:1000), Abcam; against pERK1/2 (1:1000), ERK1/2 (1:1000), CST; against Claudin19 (1:1000, Invitrogen); against Flag (1:2000, MBL International, Woburn, MD, USA); against CRALBP (1:1000), syntenin1 (1:1000), DUSP4 (1:1000), DUSP10 (1:1000), PHLPP1 (1:1000), PPM1L (1:1000), PPP1CC (1:1000), PPP2R5A (1:1000), PTPN13 (1:1000), TGF-βR2 (1:1000), and β-Actin (1:5000), Proteintech; for 12 h at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature.

Techniques: Expressing, Western Blot, Immunostaining, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration

doi: 10.1038/s41419-022-05199-5

Figure Lengend Snippet: PTPN13 binding syntenin1 in iRPE cells were identified by A mass spectrometry and confirmed by B , C Western blotting. Phosphorylation of syntenin1 was detected by D anti-phosphorylated-Tyr antibody CO-IP assay and E quantitative analysis ( n = 3). The expression levels of TGF-βR1 and TGF-βR2 in shCont-iRPE and shPtpn13-iRPE cells were detected by F Western blotting, G quantitative analysis ( n = 3). The expression levels of TGF-βR1 and TGF-βR2 on cell membranes of shCont-iRPE and shPtpn13-iRPE cells were detected by H flow cytometry and I quantitative analysis of mean fluorescence density ( n = 3). Results are expressed as mean ± SD. P value measured by Student’s unpaired t test.

Article Snippet: After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against RPE65 (1:1000, Novus Biologicals, Centennial, CO, USA); against TYRP1 (1:1000), MERTK (1:1000), FN1 (1:1000), α-SMA (1:2000), pSMAD2 (1:500), SMAD2 (1:1000), pSMAD3 (1:1000), SMAD3 (1:1000) TGF-β receptor1 (TGF-βR1) (1:1000), Abcam; against pERK1/2 (1:1000), ERK1/2 (1:1000), CST; against Claudin19 (1:1000, Invitrogen); against Flag (1:2000, MBL International, Woburn, MD, USA); against CRALBP (1:1000), syntenin1 (1:1000), DUSP4 (1:1000), DUSP10 (1:1000), PHLPP1 (1:1000), PPM1L (1:1000), PPP1CC (1:1000), PPP2R5A (1:1000), PTPN13 (1:1000), TGF-βR2 (1:1000), and β-Actin (1:5000), Proteintech; for 12 h at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature.

Techniques: Binding Assay, Mass Spectrometry, Western Blot, Phospho-proteomics, Co-Immunoprecipitation Assay, Expressing, Flow Cytometry, Fluorescence

Journal: Cell Death & Disease

Article Title: Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration

doi: 10.1038/s41419-022-05199-5

Figure Lengend Snippet: Knockdown efficiency of syntenin1 was determined by A qRT-PCR ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test), B Western blotting, and C quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). RPE-specific and EMT-associated markers in shCont-shPtpn13-iRPE and shSyntenin1-shPtpn13-iRPE cells were analyzed by D Western blotting and E quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). Phosphorylation of SMAD2/3, p38, and ERK1/2 in shCont-shPtpn13-iRPE and shSyntenin1-shPtpn13-iRPE cells were analyzed by F Western blotting and G quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). The expression levels of TGF-βR1 and TGF-βR2 on the cell membranes of shCont-shPtpn13-iRPE and shSyntenin1-shPtpn13-iRPE cells were detected by H flow cytometry and I quantitative analysis of mean fluorescence density ( n = 3, P value measured by Student’s unpaired t test). J Schematic model for the PTPN13 dephosphorylating syntenin1 mediates TGF-β receptor internalization and degradation. Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, shCont-shPtpn13-TGF-β1 compared with shSyntenin1-shPtpn13-TGF-β1; # P < 0.01, ## P < 0.01, ### P < 0.001, shCont-shPtpn13-TGF -β 2 compared with shSyntenin1-shPtpn13-TGF-β2.

Article Snippet: After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against RPE65 (1:1000, Novus Biologicals, Centennial, CO, USA); against TYRP1 (1:1000), MERTK (1:1000), FN1 (1:1000), α-SMA (1:2000), pSMAD2 (1:500), SMAD2 (1:1000), pSMAD3 (1:1000), SMAD3 (1:1000) TGF-β receptor1 (TGF-βR1) (1:1000), Abcam; against pERK1/2 (1:1000), ERK1/2 (1:1000), CST; against Claudin19 (1:1000, Invitrogen); against Flag (1:2000, MBL International, Woburn, MD, USA); against CRALBP (1:1000), syntenin1 (1:1000), DUSP4 (1:1000), DUSP10 (1:1000), PHLPP1 (1:1000), PPM1L (1:1000), PPP1CC (1:1000), PPP2R5A (1:1000), PTPN13 (1:1000), TGF-βR2 (1:1000), and β-Actin (1:5000), Proteintech; for 12 h at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Expressing, Flow Cytometry, Fluorescence

Journal: Journal of medicinal chemistry

Article Title: Imaging of Fibroblast Activation Protein in Cancer Xenografts Using Novel (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine-Based Small Molecules

doi: 10.1021/acs.jmedchem.0c02171

Figure Lengend Snippet: In vitro binding and specificity of QCP01 and [111In]QCP02. (A) Cells incubated with various concentrations (range: 50–0.78 nM) of QCP01 were imaged with the LI-COR Pearl Impulse Imager to assess binding of the agent in various FAP-positive (+) and FAP-negative (−) cell lines (left). Dose–response curves of QCP01 binding in FAP-positive cell lines (NCIH2228, U87, and SKMEL24) and FAP-negative cell lines (PC3, NCIH226, and HCT116) were generated (right). (B) Cells were incubated with 0.037 MBq [111In]QCP02 and were washed with cold phosphate-buffered saline (PBS). The radioactivity of the cell pellets was measured and normalized to the incubated dose; ****, P < 0.0001. (C) Cells incubated with 25 nM QCP01 were incubated with various concentrations of either a DPP-IV and FAP inhibitor, Val-boroPro, or a DPPIV-only inhibitor, sitagliptin. The binding of QCP01 was measured, and semi-log inhibitor–response curves were generated for both Val-boroPro (left) and sitagliptin (right).

Article Snippet: The inhibitory activities of QCP01 and [ 113/115 In] QCP02 were determined using a fluorogenic FAP Assay Kit (BPS Bioscience, San Diego, CA).

Techniques: In Vitro, Binding Assay, Incubation, Generated, Radioactivity

Journal: Journal of medicinal chemistry

Article Title: Imaging of Fibroblast Activation Protein in Cancer Xenografts Using Novel (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine-Based Small Molecules

doi: 10.1021/acs.jmedchem.0c02171

Figure Lengend Snippet: NIRF imaging of QCP01 in a tumor-bearing mouse. NOD/SCID mice bearing FAP-positive U87 (red) and FAP-negative PC3 (white) tumor xenografts (n = 4) were injected with 5 nmol of QCP01 via the tail vein, followed by serial NIRF imaging on the LI-COR Pearl Impulse Imager. Representative images of QCP01 full body (left) distribution at 5 h after injection and organ-specific (right) distribution at 5, 24, and 48 h after injection are shown.

Article Snippet: The inhibitory activities of QCP01 and [ 113/115 In] QCP02 were determined using a fluorogenic FAP Assay Kit (BPS Bioscience, San Diego, CA).

Techniques: Imaging, Injection

Journal: Journal of medicinal chemistry

Article Title: Imaging of Fibroblast Activation Protein in Cancer Xenografts Using Novel (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine-Based Small Molecules

doi: 10.1021/acs.jmedchem.0c02171

Figure Lengend Snippet: Serial SPECT-CT imaging of [111In]QCP02 in a tumor-bearing mouse. A NOD/SCID mouse bearing FAP-positive U87 (red) and FAP-negative PC3 (blue) tumor xenografts was injected with 7.4 MBq [111In]QCP02 via the tail vein, followed by serial SPECT-CT imaging. Representative three-dimensional SPECT-CT images at various time points after injection (1, 3, 6, 10, and 28 h) are shown.

Article Snippet: The inhibitory activities of QCP01 and [ 113/115 In] QCP02 were determined using a fluorogenic FAP Assay Kit (BPS Bioscience, San Diego, CA).

Techniques: Single Photon Emission Computed Tomography, Imaging, Injection

Journal: Genes & Diseases

Article Title: Cancer-associated fibroblasts derived fibronectin extra domain A promotes sorafenib resistance in hepatocellular carcinoma cells by activating SHMT1

doi: 10.1016/j.gendis.2024.101330

Figure Lengend Snippet: CAFs promote sorafenib resistance by activating NF-κB in HCC cells. (A) Immunofluorescence analysis was performed to assess the expression of FAP and α-SMA on primary CAFs. Scale bars, 50 μm. (B) Co-culture with CAFs significantly reduced apoptosis of HepG2 and Huh7 upon sorafenib treatment. (blue bar: sorafenib-treated tumor cells cultured alone; red bar: sorafenib-treated tumor cells co-cultured with CAFs). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (C) Enrichment score of the “I-κB kinase/NF-κB signaling” in sorafenib-resistant group versus sorafenib-sensitive group after sorafenib treatment, analyzed by Gene Set Enrichment Analysis based on the RNA-seq data (after performing log 2 transformation, normalization, and mean value calculation) obtained from the GEO database (GSE182593). (D) Western blot analyses of the protein level of P-p65 in the indicated HCC cells with different treatments. Results are representative of three experiments. (E) Inhibition of the NF-κB signaling pathway in HCC cells induced apoptosis significantly upon sorafenib treatment (blue bar: sorafenib-treated group; red bar: sorafenib and BAY11-7082-treated (100 μM) group). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CAFs, cancer-associated fibroblasts; NF-κB, nuclear factor kappa B; HCC, hepatocellular carcinoma; FAP, fibroblast activation protein; α-SMA, alpha-smooth muscle actin.

Article Snippet: The cells on slides were fixed with 4% paraformaldehyde for 10 min and permeabilized in phosphate buffer saline for 20 min. Then, cells were blocked with goat serum at room temperature for 60 min and incubated with primary antibodies against FAP (fibroblast activation protein; R&D system, #FAB3715A, RRID: AB_2884010) (1:200) and α-SMA (alpha-smooth muscle actin; R&D system, #MAB1420, RRID: AB_262054) (1:200) for 2 h. Then, the cells on slides were reheated and incubated with the corresponding secondary antibody at 37 °C in the dark for 2 h. Nuclei were counter-stained with DAPI.

Techniques: Immunofluorescence, Expressing, Co-Culture Assay, Cell Culture, RNA Sequencing Assay, Transformation Assay, Western Blot, Inhibition, Activation Assay