Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Transcriptomics-Based Liquid Biopsy for Early Detection of Recurrence in Locally Advanced Gastric Cancer.

doi: 10.1002/advs.202406276

Figure Lengend Snippet: Figure 2. Discovery process and preliminary validation of candidate markers for postoperative recurrence in LAGC patients based on public databases and transcriptomics sequencing data. A) Four candidate mRNAs (AGTR1, DNER, EPHA7, SUSD5) were identified through a Venn diagram analysis using the TCGA database (28 recurrent patients versus 159 non-recurrent patients), transcriptome data from the GEO database (125 recurrent patients versus 157 non-recurrent patients), and paired mRNA sequencing (3 recurrent patients versus 3 non-recurrent patients). B) A volcano plot illustrates the expression levels of these four genes in recurrent and non-recurrent cancer tissues. C) The expression levels of the four candidate mRNAs (AGTR1,

Article Snippet: Membranes were probed with antibodies against AGTR1 (1:1000 dilutions, 25343-1-AP, Proteintech, Chicago, USA), DNER (1:1000 dilutions, 24362-1-AP, Proteintech, Chicago, USA), EPHA7 (1:1000 dilutions, 66667-1-Ig, Proteintech, Chicago, USA), SUSD5 (1:500 dilutions, bs-7331R, Bioss, Beijing, China), CREB (1:500 dilutions, 381013, Zenbio, Chengdu, China), p-CREB (1:500 dilutions, 380697, Zenbio, Chengdu, China), and GAPDH (1:10000 dilutions, 10494-1-AP, Proteintech, Chicago, USA).

Techniques: Biomarker Discovery, Sequencing, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Transcriptomics-Based Liquid Biopsy for Early Detection of Recurrence in Locally Advanced Gastric Cancer.

doi: 10.1002/advs.202406276

Figure Lengend Snippet: Figure 7. Four recurrence-related mRNA genes promote GC cell proliferation, migration and invasion in vitro. A,B) Scratch assay to evaluate the migration ability of GC cells after knockdown of AGTR1 and DNER, respectively. C,D) Transwell assay to assess the invasion and metastasis abilities of GC cells after knockdown of AGTR1 and DNER, respectively. E,F) EdU assay to determine the proliferation ability of GC cells after knockdown of AGTR1 and DNER, respectively. G–J) Colony formation assay to measure the proliferation ability of GC cells after knockdown of AGTR1, DNER, EPHA7, and SUSD5. K–N) CCK-8 assay to detect the proliferation ability of GC cells after knockdown of AGTR1, DNER, EPHA7, and SUSD5. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Membranes were probed with antibodies against AGTR1 (1:1000 dilutions, 25343-1-AP, Proteintech, Chicago, USA), DNER (1:1000 dilutions, 24362-1-AP, Proteintech, Chicago, USA), EPHA7 (1:1000 dilutions, 66667-1-Ig, Proteintech, Chicago, USA), SUSD5 (1:500 dilutions, bs-7331R, Bioss, Beijing, China), CREB (1:500 dilutions, 381013, Zenbio, Chengdu, China), p-CREB (1:500 dilutions, 380697, Zenbio, Chengdu, China), and GAPDH (1:10000 dilutions, 10494-1-AP, Proteintech, Chicago, USA).

Techniques: Migration, In Vitro, Wound Healing Assay, Knockdown, Transwell Assay, EdU Assay, Colony Assay, CCK-8 Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Transcriptomics-Based Liquid Biopsy for Early Detection of Recurrence in Locally Advanced Gastric Cancer.

doi: 10.1002/advs.202406276

Figure Lengend Snippet: Figure 8. Four recurrence-related mRNA genes promote GC cell xenograft tumor growth and metastasis in vivo. A–D) Morphological images showing reduced subcutaneous xenograft tumor formation in mice injected with AGS cells knocked down for AGTR1, DNER, EPHA7, and SUSD5, along with tumor volume growth curves and final tumor weights. E) Representative IHC images of subcutaneous xenograft tumors after knockdown of AGTR1 (left), and quantification of IHC staining data for Ki67, N-cadherin, E-cadherin, and Vimentin in each group of mice (right). F) Representative IHC images of peritoneal metastasis tumors after intraperitoneal injection of AGS cells knocked down for AGTR1 (left), and quantification of IHC staining data for MMP9, N-cadherin, E-cadherin, and Vimentin in each group of mice (right). G) Representative images of peritoneal metastasis tumors in the abdominal cavity of mice injected with AGS cells knocked down for AGTR1. H) Measurement and quantification of the number of peritoneal metastatic tumors in

Article Snippet: Membranes were probed with antibodies against AGTR1 (1:1000 dilutions, 25343-1-AP, Proteintech, Chicago, USA), DNER (1:1000 dilutions, 24362-1-AP, Proteintech, Chicago, USA), EPHA7 (1:1000 dilutions, 66667-1-Ig, Proteintech, Chicago, USA), SUSD5 (1:500 dilutions, bs-7331R, Bioss, Beijing, China), CREB (1:500 dilutions, 381013, Zenbio, Chengdu, China), p-CREB (1:500 dilutions, 380697, Zenbio, Chengdu, China), and GAPDH (1:10000 dilutions, 10494-1-AP, Proteintech, Chicago, USA).

Techniques: In Vivo, Injection, Knockdown, Immunohistochemistry

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression.

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: Fig. 1 P4HA2 interacts with KIF7 and regulates the Hedgehog signaling. A P4HA2 interacts with KIF7. HEK293T cells were transfected with Flag-tagged P4HA2. Cell lysates were prepared and subjected to immunoprecipitation (IP) with M2 anti-Flag beads. The immunoprecipitates were analyzed by Western blotting (WB) with anti-Flag and KIF7 antibodies. B KIF7 interacts with P4HA2. HEK293T cells were transfected with Flag-tagged KIF7. Cell lysates were prepared and subjected to Flag IP. The immunoprecipitants were analyzed by WB with anti-Flag and P4HA2 antibodies. C Schematic representation of the SHH signaling pathway. SAG is a chlorobenzothiophene-containing Hh pathway agonist and binds to the SMO heptahelical bundle in a manner that antagonizes cyclopamine action. SAG activates SMO, leading to the release of GLI1 by SUFU, and the Hh signaling pathway is then activated. D–I Knockout of P4HA2 causes suppression of Hedgehog signal transduction. NIH/3T3 (D–F) and OP9 (G–I) P4HA2 knockout (KO) or vehicle cells were treated with (+) or without (−) 200 nM SAG. The Hh targeted genes, Gli1 (E, H) and Ptch1 (F, I), were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Data are shown as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All experiments were repeated three times independently.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF): PAN-OH (home-made; 1:1,000 for WB); P4HA2 (13759-1- AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC, 1:200 for IF); P4HA1 (12658-1-AP, rabbit, Proteintech; 1:1,000 for WB); KIF7 (24693-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC); SUFU (26759-1-AP, rabbit, Proteintech; 1:2,000 for WB); Flag (clone M2, F1804, mouse, Sigma-Aldrich; 1:10,000 for WB, 1:200 for IF); His (clone OGHis, D291-3, mouse, Medical & Biological Laboratories; 1:5,000 for WB); V5 (AB3792, rabbit, Merck Millipore; 1:2,000 for WB); GLI1 (clone A-7, sc-515781, mouse, Santa Cruz; 1:1,000 for WB, 1:200 for IHC); GLI2 (18989-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC) ; GAPDH (clone 1E6D9, 60004-1-Ig, mouse, Proteintech; 1:10,000 for WB); Arl13b (clone N295B/66, 75-287, mouse, NeuroMab; 1:200 for IF); Cy3 AffiniPure Goat Anti-Mouse IgG (H+ L) (115- 165-003, Jackson ImmunoResearch; 1:10,000 for IF); α-SMA (#34105, Cell Signaling Technology, 1:100 for IF) Light chain specific Mouse Anti-Rabbit IgG (211-032-171, Jackson ImmunoResearch; 1:10,000 for WB); Light chain specific Goat Anti Mouse IgG (115-035-174, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific (111-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Donkey anti-mouse IgG (H&L) (715-035-150, Jackson ImmunoResearch; 1:10,000 for WB); Goat Anti-Rabbit IgG (H+ L) (111-035-003, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (115-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Smoothened Agonist (SAG), Selleck, Cat# S7779; Fetal bovine serum, Thermo Fisher, Cat# 16140071; Penicillin/streptomycin, Thermo Fisher, Cat# 15140122; RPMI 1640, Thermo Fisher, Cat# 11875093; DMEM, Thermo Fisher, Cat# 11995065; Puromycin Dihydrochloride, Sigma, Cat# P8833; Trypsin 0.25% EDTA, Thermo Fisher, Cat# 25200072; Polyethylenimine Hydrochloride (MW 40,000), Polysciences, Cat# 24765-1; Polybrene Transfection Reagent, Sigma, Cat# TR-1003-G; SHH protein, Yun Zhao Laboratory; Anti-V5-tag mAb-Magnetic Beads, Medical & Biological Laboratories, Cat# M215-11; NTI-FLAG® M2 Affinity Gel, Sigma, Cat# F2426; Ni-NTA Agarose, QIAGEN, Cat# 30230; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01/02; ChamQ SYBR qPCR Master Mix, Vazyme, Cat# Q311-02.

Techniques: Transfection, Immunoprecipitation, Western Blot, Knock-Out, Transduction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression.

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: Fig. 3 P4HA2 migrates with KIF7 to regulate the Hedgehog pathway transduction. A The trafficking of the P4HA2-KIF7 complex from the cytoplasm to the cilium responds to Hedgehog signaling. NIH/3T3 cells were co-infected with KIF7-BFP and P4HA2-EGFP lentivirus. White arrow indicates the co-localization of cilia, KIF7-BFP and P4HA2-EGFP. Yellow arrow indicates the co-localization of cilia and P4HA2-EGFP, which may bind with endogenous KIF7. Cells were treated with 200 nM SAG (+) or not (−). Cells were fixed and stained with antibodies for ARL13B to mark primary cilia. Scale bars: 5 μm. B Statistical analysis of the relative colocalization rate in (A) indicated cells. Data are shown as the mean ± SEM; SAG (−), n = 11; SAG (+), n = 13. **P < 0.01. C The regulation of the Hedgehog signaling by P4ha2 is dependent on KIF7. KIF7 knockout (KO) and the control cells were knocked down with P4HA2 (shP4HA2). Cells were treated with (+) or without (−) 200 nM SAG. qRT- PCR analysis of GLI1 transcripts was performed using RNA isolated from the indicated cell lines. Data are shown as the mean ± SEM (n = 3). ***P < 0.001. All experiments were repeated three times independently.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF): PAN-OH (home-made; 1:1,000 for WB); P4HA2 (13759-1- AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC, 1:200 for IF); P4HA1 (12658-1-AP, rabbit, Proteintech; 1:1,000 for WB); KIF7 (24693-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC); SUFU (26759-1-AP, rabbit, Proteintech; 1:2,000 for WB); Flag (clone M2, F1804, mouse, Sigma-Aldrich; 1:10,000 for WB, 1:200 for IF); His (clone OGHis, D291-3, mouse, Medical & Biological Laboratories; 1:5,000 for WB); V5 (AB3792, rabbit, Merck Millipore; 1:2,000 for WB); GLI1 (clone A-7, sc-515781, mouse, Santa Cruz; 1:1,000 for WB, 1:200 for IHC); GLI2 (18989-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC) ; GAPDH (clone 1E6D9, 60004-1-Ig, mouse, Proteintech; 1:10,000 for WB); Arl13b (clone N295B/66, 75-287, mouse, NeuroMab; 1:200 for IF); Cy3 AffiniPure Goat Anti-Mouse IgG (H+ L) (115- 165-003, Jackson ImmunoResearch; 1:10,000 for IF); α-SMA (#34105, Cell Signaling Technology, 1:100 for IF) Light chain specific Mouse Anti-Rabbit IgG (211-032-171, Jackson ImmunoResearch; 1:10,000 for WB); Light chain specific Goat Anti Mouse IgG (115-035-174, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific (111-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Donkey anti-mouse IgG (H&L) (715-035-150, Jackson ImmunoResearch; 1:10,000 for WB); Goat Anti-Rabbit IgG (H+ L) (111-035-003, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (115-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Smoothened Agonist (SAG), Selleck, Cat# S7779; Fetal bovine serum, Thermo Fisher, Cat# 16140071; Penicillin/streptomycin, Thermo Fisher, Cat# 15140122; RPMI 1640, Thermo Fisher, Cat# 11875093; DMEM, Thermo Fisher, Cat# 11995065; Puromycin Dihydrochloride, Sigma, Cat# P8833; Trypsin 0.25% EDTA, Thermo Fisher, Cat# 25200072; Polyethylenimine Hydrochloride (MW 40,000), Polysciences, Cat# 24765-1; Polybrene Transfection Reagent, Sigma, Cat# TR-1003-G; SHH protein, Yun Zhao Laboratory; Anti-V5-tag mAb-Magnetic Beads, Medical & Biological Laboratories, Cat# M215-11; NTI-FLAG® M2 Affinity Gel, Sigma, Cat# F2426; Ni-NTA Agarose, QIAGEN, Cat# 30230; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01/02; ChamQ SYBR qPCR Master Mix, Vazyme, Cat# Q311-02.

Techniques: Transduction, Infection, Staining, Knock-Out, Control, Quantitative RT-PCR, Isolation