ags human gc cell line  (ATCC)

Journal: Nucleic Acids Research

Article Title: A novel dual histone mark reader ZCWPW2 regulates meiotic recombination through lactylation and transcriptional regulation in humans and mice

doi: 10.1093/nar/gkag049

Figure Lengend Snippet: The ZCWPW1–ZCWPW2 complex interacts with recombination-associated proteins and stabilizes LDHA activity. ( A ) Schematic of IP-MS-based identification of ZCWPW2-interacting proteins in human testis tissue. ( B ) GO enrichment analysis of ZCWPW2-interacting proteins. ( C ) Mass spectrometry–based intensity plot depicting meiosis-associated proteins that interact with ZCWPW2. ( D and E ) Co-IP analysis of endogenous SYCP1, TEX11, HSPA2, MLH1, and MSH2 proteins interacting with ZCWPW2 from human (D) and mouse (E) testis lysates. ( F ) Co-IP analysis of exogenous SYCP1, TEX11, HSPA2, MLH1, and MSH2 proteins interacting with exogenous ZCWPW2 in HEK293T cells. ( G–L ) Co-IP assays demonstrating the impact of ZCWPW1 knockdown (siRNA- W1 ) or overexpression (Myc- W1 ) on the interactions of Flag-ZCWPW2 with HA-HSPA2 (G), SYCP1 (H), MLH1 (I), MSH2 (J), TEX11 (K), and MDC1 (L) in HEK293T cells. ( M ) Co-IP assays demonstrating the impact of Zcwpw1 knockdown (siRNA- w1 ) on the interactions of endogenous Zcwpw2 with Hspa2, Sycp1, Mlh1, Msh2, Tex11, Mdc1, and Ldha in GC-2 cells. ( N ) Table summarizing the LDH family proteins detected in ZCWPW2 immunoprecipitates from human testis by IP-MS. ( O ) Co-IP assays demonstrating the interaction between ZCWPW2 and LDHA in human and mouse testes, as well as transfected HEK293T cells. ( P ) Co-IP analysis examining the interaction between Flag-ZCWPW2 and Myc-LDHA in HEK293T cells under conditions of ZCWPW1 knockdown (siRNA- W1 ) or overexpression (HA- W1 ). ( Q ) Schematic overview (top) and quantitative analysis (bottom) of LDH enzymatic activity in testis lysates from WT and Zcwpw2 KO juvenile mice. Activity was measured by assessing formazan absorbance at 450 nm following the conversion of lactate using the WST-8 assay ( n = 3 biologically independent WT mice and KO mice; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( R and S ) LDH enzymatic activity measured by WST-8-based formazan absorbance in treated GC-2 cells (R) and HEK293T cells (S) ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.).

Article Snippet: HEK293T cells (CRL-11268; ATCC) and GC-2 cells (CRL-2196; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS; Gibco, 12483020).

Techniques: Activity Assay, Protein-Protein interactions, Mass Spectrometry, Co-Immunoprecipitation Assay, Knockdown, Over Expression, Transfection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: A novel dual histone mark reader ZCWPW2 regulates meiotic recombination through lactylation and transcriptional regulation in humans and mice

doi: 10.1093/nar/gkag049

Figure Lengend Snippet: ZCWPW2 promotes lactylation and stability of recombination-associated proteins through coordination with ZCWPW1 in cultured cells. ( A ) Co-IP analysis of HEK293T cells overexpressed with HA-recombination-associated proteins (HSPA2, SYCP1, and MDC1) and Flag-ZCWPW2, with or without Myc-ZCWPW1, and in the presence or absence of siRNA targeting ZCWPW1 . Bar graph quantifies Kla signal intensity for each condition ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( B ) Co-IP analysis (left) for Kla of endogenous Mdc1, Hspa2, and Sycp1 in GC-2 cells after individual or combined knockdown of Zcwpw1 or/and Zcwpw2 using siRNAs. Quantification of Kla levels (right) showed a significant decrease in Kla upon Zcwpw1 or Zcwpw2 knockdown, with the greatest reduction observed upon dual silencing ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( C and D ) Co-IP (left) and quantification analysis (right) of Kla on endogenous Mdc1, Hspa2, and Sycp1 in GC-2 cells treated with the EP300 inhibitor A-485, the LDHA inhibitor sodium oxamate, or both (C), and with EP300 activator CTPB or exogenous lactate (D) ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.).

Article Snippet: HEK293T cells (CRL-11268; ATCC) and GC-2 cells (CRL-2196; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS; Gibco, 12483020).

Techniques: Cell Culture, Co-Immunoprecipitation Assay, Two Tailed Test, Knockdown

Journal: Frontiers in Genetics

Article Title: Systematic analysis of anoikis-related genes identifies SRPX2-FAK/AKT-IL-6 axis in the progression and peritoneal metastasis of gastric cancer

doi: 10.3389/fgene.2025.1736097

Figure Lengend Snippet: SRPX2 in CAFs promotes malignant GC progression in vitro and in vivo . (A,B) Transwell migration assay and quantitative analysis of HGC and AGS cells transfected with siSRPX2#1 or siSRPX2#2. (C) Immunofluorescence staining showing subcellular localization and expression of α-SMA in isolated CAFs and NFs. (D) qRT-PCR analysis of SRPX2 expression levels in isolated CAFs and NFs. (E,F) Colony formation assay of HGC and AGS cells co-cultured with PBS, NFs, or CAFs. (G,H) Transwell migration assay of HGC and AGS cells co-cultured with PBS, NFs, or CAFs. (I) Wound healing assay of HGC and AGS cells co-cultured with PBS, NFs, or CAFs. (J) Flow cytometry analysis of apoptosis in AGS cells under anoikis conditions (low-attachment culture) after co-culture with PBS, NFs, or CAFs. (K–O) Colony formation, Transwell migration, and wound healing assays of HGC and AGS cells cultured with conditioned medium from CAFs transfected with siSRPX2#1 or siSRPX2#2. (P) Flow cytometry analysis of apoptosis rates in AGS cells cultured with conditioned medium from CAFs transfected with siSRPX2#1 or siSRPX2#2. (Q–T) In vivo xenograft tumor assay: (Q) representative images of tumors from mice co-injected with MKN45 cells and CAFs (n = 5) or MKN45 cells and SRPX2-knockdown CAFs (shSRPX2, n = 5); (R) tumor growth curves; (S) average tumor weight; (T) representative H&E staining and Ki-67 immunohistochemistry of xenograft tumor sections. All data are presented as mean ± SD of triplicate experiments. ns, P > 0.05; ***, P < 0.001.

Article Snippet: The GC cell lines AGS, HGC-27, and MKN45 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, United States) and maintained in the Gastrointestinal Oncology Laboratory at Sun Yat-sen Memorial Hospital, Sun Yat-sen University.

Techniques: In Vitro, In Vivo, Transwell Migration Assay, Transfection, Immunofluorescence, Staining, Expressing, Isolation, Quantitative RT-PCR, Colony Assay, Cell Culture, Wound Healing Assay, Flow Cytometry, Co-Culture Assay, Migration, Injection, Knockdown, Immunohistochemistry

Journal: Frontiers in Genetics

Article Title: Systematic analysis of anoikis-related genes identifies SRPX2-FAK/AKT-IL-6 axis in the progression and peritoneal metastasis of gastric cancer

doi: 10.3389/fgene.2025.1736097

Figure Lengend Snippet: SRPX2 in CAFs activates the FAK/AKT pathway and secretes IL-6 to induce anoikis resistance in GC. (A) Western blot analysis of SRPX2, p-FAK, FAK, p-AKT, and AKT expression in CAFs transfected with siSRPX2#1 or siSRPX2#2. (B) Expression levels of p-FAK and p-AKT in CAFs overexpressing SRPX2 and treated with the FAK pathway inhibitor PF-573228 (5 μM). (C) Expression of SRPX2, p-FAK, and p-AKT in CAFs overexpressing TFAP2A, followed by SRPX2 knockdown. (D,E) Relative IL-6 mRNA expression in CAFs after SRPX2 knockdown or overexpression, measured by qRT-PCR. (F,G) IL-6 secretion levels in conditioned medium from CAFs after SRPX2 knockdown or overexpression, as determined by ELISA. (H) Apoptosis analysis by Annexin V/PI staining in AGS cells cultured under anoikis conditions (low-attachment culture) after treatment with conditioned medium from SRPX2-overexpressing CAFs pretreated with tocilizumab (10 μg/mL) for 24 h. (I,J) Tocilizumab treatment significantly suppressed subcutaneous tumor growth in BALB/c nude mice injected with MKN45 cells and SRPX2-overexpressing CAFs. (K) In vivo monitoring of GC cell dissemination in the abdominal cavity using IVIS imaging; tocilizumab treatment markedly inhibited peritoneal metastasis. (L) Representative images of peritoneal metastases in mice at the end of the study. All data are presented as mean ± SD of triplicate experiments. ***, P < 0.001.

Article Snippet: The GC cell lines AGS, HGC-27, and MKN45 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, United States) and maintained in the Gastrointestinal Oncology Laboratory at Sun Yat-sen Memorial Hospital, Sun Yat-sen University.

Techniques: Western Blot, Expressing, Transfection, Knockdown, Over Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Cell Culture, Injection, In Vivo, Imaging

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Effects of melatonin and agomelatine on proliferation loss in GC-1 spermatogonia (spg) treated with ivermectin (IVM) for 24 h (A) Chemical structure of IVM isomers. (B) Chemical structures of melatonin and its analogs, agomelatine and pinoline. (C) Schematic overview of the cell culture and experimental design. (D) Brightfield microscopy images showing cytoplasmic morphology. (E) Immunofluorescence images of Ki67 nuclear translocation. (F) Quantification of relative cell proliferation (n = 3). (G) Quantification of Ki67-positive nuclei (n = 3) in GC-1 spg treated with either control, melatonin, agomelatine, or pinoline with or without 16 μM IVM. IVM treatment conditions are denoted as positive (+) or negative (−). Scale bars: 100 µm. Data are presented as means ± SEM. Significant differences are indicated by different letters (a–d) at p < 0.05.

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques: Cell Culture, Microscopy, Immunofluorescence, Translocation Assay, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Inhibition of intracellular reactive oxygen species (ROS) generation by melatonin and agomelatine in GC-1 spermatogonia (spg) treated with ivermectin (IVM) for 3 h (A) Fluorescence microscopy images of GC-1 spg labeled with DCFDA. (B) Semi-quantification of DCFDA fluorescence intensity in GC-1 spg treated with either control, melatonin, agomelatine, or pinoline with or without 16 μM IVM (n = 5). IVM treatment conditions are denoted as positive (+) or negative (−). Scale bars: 100 µm. Data are shown as means ± SEM. Significant differences are indicated by different letters (a–d) at p < 0.05. Mel, melatonin; Ago, agomelatine; Pin, pinoline.

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques: Inhibition, Fluorescence, Microscopy, Labeling, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Melatonin and agomelatine inhibit cytoplasmic Ca 2+ accumulation in GC-1 spermatogonia (spg) treated with ivermectin (IVM) for 3 h (A) Fluorescence microscopy images of GC-1 spg cells labeled with Fluo-4, AM. (B) Semi-quantification of Fluo-4, AM fluorescence intensity in GC-1 spg treated with either control, melatonin, agomelatine, or pinoline with or without 16 μM IVM (n = 5). IVM treatment conditions are denoted as positive (+) or negative (−). Scale bars: 100 µm. Data are presented as means ± SEM. Significant differences are denoted by different letters (a–c) at p < 0.05. Mel, melatonin; Ago, agomelatine; Pin, pinoline.

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques: Fluorescence, Microscopy, Labeling, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Melatonin’s and agomelatine’s recovery of ΔΨm and mitochondrial mass in GC-1 spermatogonia (spg) treated with 16 μM ivermectin (IVM) for 3 h (A) Fluorescence microscopy images of GC-1 spg stained with tetramethylrhodamine ethyl ester (TMRE) and/or MitoTracker and Hoechst 33,342. (B) Semi-quantification of MitoTracker fluorescence intensity (n = 5), and (C) ratio of TMRE to MitoTracker fluorescence intensity in GC-1 spg treated with either control, melatonin, agomelatine, or pinoline with or without 16 μM IVM (n = 5). Scale bars: 100 µm. IVM treatment conditions are denoted as positive (+) or negative (−). Data are shown as means ± SEM. Significant differences are denoted by different letters (a–c) at p < 0.05. Mel, melatonin; Ago, agomelatine; Pin, pinoline

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques: Fluorescence, Microscopy, Staining, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Seahorse assay of GC-1 spermatogonia (spg) treated with or without melatonin analogs and/or ivermectin (IVM) for 3 h (A) Oxygen consumption rate (OCR) plot following sequential injections of oligomycin, FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), and rotenone/antimycin A (R/AA). (B) Basal respiration, (C) ATP-linked respiration, (D) maximal respiration capacity, and (E) reserve capacity calculated from OCR data (n = 3). Data are presented as means ± SEM. Significant differences are indicated by different letters (a–d) at p < 0.05. Mel, melatonin; Ago, agomelatine; Pin, pinoline.

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Attenuation of apoptosis by melatonin and agomelatine in GC-1 spermatogonia (spg) treated with ivermectin (IVM) for 3 h (A) Brightfield microscopy images showing morphological changes in GC-1 spg following treatment with IVM and/or melatonin analogs. Scale bars: 100 µm. (B) Western blot analysis of apoptosis-related proteins, including cleaved caspases, BCL-2, BAX, Cytochrome c, and α-tubulin (used as a loading control), under the indicated treatment conditions. Mel, melatonin; Ago, agomelatine; Pin, pinoline (C) Quantification of apoptosis-related proteins (n = 3). Data are shown as means ± SEM. Significant differences are denoted by different letters (a–c) at p < 0.05. Mel, melatonin; Ago, agomelatine; Pin, pinoline.

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques: Microscopy, Western Blot, Control