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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 1. Expression and distribution of Lgals3 in both mouse and human CaOx stone specimens. A) heatmap for differentially expressed Lgals family member in the kidney tissues from NC mice and CaOx stone mice based on the RNA-seq. B) heatmap for differentially expressed Lgals family member in the kidney tissues from NC mice and CaOx stone mice based on the DIA proteomic analysis. C) The expression of Lgals3 based on the result of RNA-seq. D) The expression of Lgals3 based on the DIA proteomic analysis. E-F) The immunofluorescence images and quantification of Lgals3 and 𝛼-SMA levels in the kidney tissues from NC mice and CaOx stone mice (scale bar = 100 μm in first colcumn and 40 μm in second colcums, n = 5 mice per group). G) Immunoblots of the protein expression levels of Lgals3 in kidney tissues from NC mice and CaOx stone mice (n = 5 mice per group). H) Immunoblots of the protein expression levels of Lgals3 in HK-2 cells treated with COM for 48h. I) Immunofluorescence images showing the Lgals3 level in HK-2 cell with COM treated for 48h. J) Immunoblots of the protein expression levels of Lgals3 in kidney tissues from CaOx kidney stone patients (n = 10 per group). K) mRNA levels of Lgals1-Lgals7 in kidney tissues from CaOx kidney stone patients (n = 10 per group). L,M) Immunohistochemical staining and quantification of Lgals3 in kidney tissues from CaOx kidney stone patients. (scale bar = 50 μm, n = 10 per group). N) The correlation between Lgals3 expression and kidney function (eGFR) in the clinical cohort. *P<0.01; **P<0.05.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Expressing, RNA Sequencing, Western Blot, Immunohistochemical staining, Staining

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 2. Lgals3 deficiency inhibits kidney injury and renal fibrosis caused by CaOx crystal. A) The schematic of the experimental design. B-C) The BUN and Scr level in blood from WT mice and Lgals3 knockout (Lgals3−/−) mice (n = 5 mice per group). D) Representative images and quantification of HE and Von-kossa staining in kidney tissues from WT mice and Lgals3 knockout (Lgals3−/−) mice (scale bar = 50 μm, n = 5 mice per group). E) Representative images and quantification of Masson staining and the immunohistochemical staining of 𝛼-SMA and Collagen1 in kidney tissues from WT mice and Lgals3 knockout (Lgals3−/−) mice (scale bar = 50 μm, n = 5 mice per group). F) Immunoblots of the protein expression levels and quantification of Fibronectin, 𝛼-SMA and Collagen1 in kidney tissues from WT mice and Lgals3 knockout (Lgals3−/−) mice (n = 5 mice per group). **P<0.01, compared to the NC group; #P<0.05, compared with WT-Stone mice.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Knock-Out, Staining, Immunohistochemical staining, Western Blot, Expressing

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 3. Lgals3 overexpression promotes kidney injury and renal fibrosis induced by CaOx crystal. A) The schematic of the experimental design. B,C) The BUN and Scr level in blood from AAV9-NC mice and AAV9-Lgals3 mice (n = 5 mice per group). D) Representative images and quantification of HE and Von-kossa staining in kidney tissues from AAV9-NC mice and AAV9-Lgals3 mice (scale bar = 50 um, n = 5 mice per group). E) Representative images and quantification of Masson staining and the immunohistochemical staining of 𝛼-SMA and Collagen1 in kidney tissues from AAV9-NC mice and AAV9-Lgals3 mice (scale bar = 50 um, n = 5 mice per group). F) Immunoblots of the protein expression levels and quantification of Fibronectin, 𝛼-SMA and Collagen1 in kidney tissues from AAV9-NC mice and AAV9-Lgals3 mice (n = 5 mice per group). **P<0.01, compared to the NC group; #P<0.05, compared with AAV9-Ctrl-Stone mice.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Over Expression, Staining, Immunohistochemical staining, Western Blot, Expressing

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 4. Lgals3 regulates glycolysis during the process of kidney injury caused by CaOx crystal. A) Volcano plot for differentially expressed genes (DEGs) in the kidney tissues from WT mice and Lgals3 knockout (Lgals3−/−) mice that with CaOx crystal deposition. (n = 5 mice per group). B) Bubble chart showing the KEGG pathway enrichment analysis of the DEGs. C) Gene Set Enrichment Analysis (GSEA) of WT and Lgals3−/−mice from RNA-seq data. D) Volcano plot for differentially expressed proteins (DEPs) in the kidney tissues from WT mice and Lgals3 knockout (Lgals3−/−) mice (n = 5 mice per group). E) Bubble chart showing the KEGG pathway enrichment analysis of the DEPs. F) Gene Set Enrichment Analysis (GSEA) of WT and Lgals3−/−

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Knock-Out, RNA Sequencing

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 5. Lgals3 interacts with PKM2 and regulates PKM2 level to promote lactate generation. A) The schedule of IP-MS. B) Silver staining showing IP products. C) The top8 proteins that binding to Lgals3. D) The overlap protein from the IP-MS database and DIA protemic analysis. E) The peptide of PKM2 from IP-MS. F) The molecular docking of Lgals3 and PKM2. G) Cell lysates of HK-2 cells were immunoprecipitated with Lgals3 or PKM2 antibodies, and immunoblot assays were performed. H) 293T cells were transfected with plasmids encoding Flag-Lgals3 or His-PKM2. Cell lysates were immunoprecipitated with Flag and HA antibodies and immunoblot assays were performed. I) HK-2 cells were transfected with plasmids encoding Flag- Lgals3 or His-PKM2. Cell lysates were immunoprecipitated with Flag and HA antibodies and immunoblot assays were performed. J) Representative images of the immunofluorescence staining of Lgals3 (green) and PKM2 (red) in kidney tissues. K) Co-localization of Lgals3 (green) and PKM2 (red) in the HK-2 cells.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Protein-Protein interactions, Silver Staining, Binding Assay, Immunoprecipitation, Western Blot, Transfection, Staining

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 6. Lgals3 inhibits PKM2 Ubiquitination and degradation. A) Immunoblots of the protein expression levels of PKM2 in HK-2 and 293T cells transfected with the indicated doses of plasmids encoding Flag-Lgals3. **P<0.01, ***P<0.005, compared to the NC group. B) The mRNA level of PKM2 in HK-2 and 293T cells transfected with the indicated doses of plasmids encoding Flag-Lgals3. C) Cycloheximide (CHX) was used to block protein synthesis. Immunoblots of the protein expression levels and quantification of PKM2 in HK-2 cells. D) Cycloheximide (CHX) was used to block protein synthesis. Immunoblots of the protein expression levels and quantification of PKM2 in 293T cells. E) Immunoblots of the protein expression levels and quantification of PKM2 in MG132 treated HK-2 cells (n = 3 per group). F) Immunoblots of the protein expression levels and quantification of PKM2 in CQ and NH4CL treated HK-2 cells (n = 3 per group). **P<0.01, compared to the Sh-Ctrl group; #P<0.05, compared with Sh-Lgals3 group. G) Detection of endogenous ubiquitination levels of PKM2 in COM treated HK-2 cells. H) Detection of endogenous ubiquitination levels of PKM2 in kidney tissues of CaOx mice. I,J) Cells were pretreated with MG132 (5 um) for 8h. Detection of the ubiquitinated levels of PKM2 in Lgals3-overexpression or Lgals3-knockdown HK-2 cells.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Transfection, Blocking Assay, Over Expression, Knockdown

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 7. Lgals3 inhibits PKM2 Ubiquitination and degradation by interact with Trim21. A-D) Cell lysates of HK-2 cells were immunoprecipitated with Flag, Myc and His antibodies, and immunoblot assays were performed. E) Immunoblots of the protein expression levels of PKM2 in HK-2 and 293T cells transfected with the indicated doses of plasmids encoding Myc-Trim21. F) The mRNA level of PKM2 in HK-2 and 293T cells transfected with the indicated doses of plasmids encoding Myc-Trim21. G) Immunoblots of the protein expression levels and quantification of PKM2 in Trim21 knockdown HK-2 cells (n = 3 per group). H) Cycloheximide (CHX) was used to block protein synthesis. Immunoblots of the protein expression levels and quantification of PKM2 in HK-2 cells. I) Cycloheximide (CHX) was used to block protein synthesis. Immunoblots of the protein expression levels and quantification of PKM2 in 293T cells. J) Immunoblots of the protein expression levels and quantification of PKM2 in MG132 treated HK-2 cells (n = 3 per group). **P<0.01, compared to the Myc-Ctrl group; #P<0.05, compared with Myc-Trim21 group. K) Immunoblots of the protein expression levels and quantification of PKM2 in CQ and NH4CL treated HK-2 cells (n = 3 per group). L-M) HK-2 and 293T cells were pretreated with MG132 (5 um) for 8h. Detection of the ubiquitinated levels of PKM2 in Trim21-overexpression or Trim21-knockdown cells. N) Co-localization of Lgals3, PKM2 and Trim21 in HK-2 cells.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing, Transfection, Knockdown, Blocking Assay, Over Expression

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 9. Inhibition of Lgals3 reduces H3K18la during the formation of CaOx crystal. A) The schematic of the experimental design. B) Representative images and quantification of the von-kossa staining and the immunofluorescence staining of 𝛼-SMA in kidney tissues from mice treated with FX-11 (scale bar = 50 um, n = 5 mice per group). **P<0.01, compared to the Stone group. C) Immunoblots of the protein expression levels and quantification of H3K18la in HK-2 cells with FX-11 treated for 48h (n = 3 per group). D) Immunofluorescence images showing the 𝛼-SMA level in HK-2 cell with FX-11 treated for 48h (n = 3 per group). E) Immunoblots of the protein expression levels and quantification of Lacty-lysine in Lgals3 knockdown HK-2 cells with COM treated for 48h (n = 3 per group). F) Immunoblots of the protein expression levels and quantification of H3K9la, H3K14la, H3K18la and H3K56la in Lgals3 knockdown HK-2 cells with COM treated for 48h (n = 3 per group). G) Immunofluorescence images showing the H3K18la level in Lgals3 knockdown HK-2 cell with COM treated for 48h (n = 3 per group). **P<0.01, compared to the COM group. H) Representative images and quantification of the immunofluorescence staining of Lacty-lysine and 𝛼-SMA in kidney tissues from WT mice and Lgals3 knockout mice (scale bar = 50 um, n = 5 mice per group). I) Representative images and quantification of the immunofluorescence staining of H3K18la and 𝛼-SMA in kidney tissues from WT mice and Lgals3 knockout mice (scale bar = 50 um, n = 5 mice per group). **P<0.01, compared to the WT+Stone group. J) Immunoblots of the protein expression levels and quantification of Lacty-lysine and H3K18la in kidney tissues from WT mice and Lgals3 knockout (Lgals3−/−) mice (n = 5 mice per group). **P<0.01, compared to the NC group; #P<0.05, compared with WT-Stone mice.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Inhibition, Staining, Western Blot, Expressing, Knockdown, Knock-Out

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 10. Lgals3-mediated H3K18la targets multiple fibrosis-related genes. A,B) The binding density of H3K18la in the transcriptional start site was visualized. C) Genome-wide distribution of differentiated H4K12la-binding peaks in Sh-Ctrl HK-2 cell and Sh-Lgals3 HK-2 cells. D) Volcano plot for different genetic loci bound by H3K18la in CUT-tag. E) Bubble chart showing the KEGG pathway enrichment analysis of the H3K18la binding DEGs. F) Venn diagram showed genes downregulated in WT and Lgals3 knockout mice and the downregulated target genes bound by H3K18la. G) Integrative Genomics Viewer analysis representing H3K18la peaks at the FGFR4 locus. H) Chip-qPCR assays of H3K18la in the FGFR4 in HK-2 cells. I) Immunoblots of the protein expression levels and quantification of FGFR4 in kidney tissues from WT mice and Lgals3 knockout (Lgals3−/−) mice (n = 5 mice per group). **P<0.01, compared to the NC group; #P<0.05, compared with WT-Stone mice. J) Immunoblots of the protein expression levels and quantification of FGFR4 in Lgals3 knockdown HK-2 cells with COM treated for 48h (n = 3 per group). **P<0.01, compared to the Sh-Ctrl group; #P<0.05, compared with COM+Sh-Ctrl group. K) Immunofluorescence images showing the 𝛼-SMA level in Lgals3 overexpression HK-2 cells with BLU9931 treated for 48h (n = 3 per group). L) Immunoblots of the protein expression levels and quantification of Collagen1 and 𝛼-SMA in Lgals3 overexpression HK-2 cells with BLU9931 treated for 48h (n = 3 per group). **P<0.01, compared to the Lv-Vector group; #P<0.05, compared with Lv-Lgals3 group.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Binding Assay, Genome Wide, Knock-Out, ChIP-qPCR, Western Blot, Expressing, Knockdown, Over Expression, Plasmid Preparation

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 11. Pharmacological inhibition of Lgals3 ameliorates CaOx crystal formation and renal fibrosis. A) The schematic of the experimental design. B) The BUN and Scr level in blood from Caox stone mice and CaOx stone+MCP mice (n = 5 mice per group). C,D) Representative images and quantification of HE, Von-kossa and 𝛼-SMA staining in kidney tissues from Caox stone mice and CaOx stone+MCP mice (scale bar = 50 um, n = 5 mice per group). E,F) Immunoblots of the protein expression levels and quantification of PKM2, Lacty-lysine, H3K18la, FGFR4, 𝛼-SMA and Collagen1in kidney tissues from Caox stone mice and CaOx stone+MCP mice (n = 5 mice per group). **P<0.01, compared to the NC group; #P<0.05, compared with Stone group.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Inhibition, Staining, Western Blot, Expressing

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 12. Lgals3 may be key factors involved in CaOx stone patients. A–C) Graphic presentation shows serum Lgals3 levels in a cohort of patients with nephrolithiasis (n = 10) and healthy participants (n = 10). D,E) The H3K18la level in the kidney tissues from CaOx stone patients. **P<0.01 compared to the NC group.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques:

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

Article Title: Lgals3 Promotes Calcium Oxalate Crystal Formation and Kidney Injury Through Histone Lactylation-Mediated FGFR4 Activation.

doi: 10.1002/advs.202413937

Figure Lengend Snippet: Figure 13. Schematic representation of the mechanism of Lgals3 in CaOx stone formation. Elevated Lgals3 interacted with PKM2 and promoted the expression of FGFR4 via H3K18la, thereby facilitating CaOx crystal deposition and the development of renal fibrosis.

Article Snippet: Lgals3 knockout mice (Lgals3−/− mice) were obtained from the Cyagen (Suzou, China).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Distinct cholangiocarcinoma cell migration in 2D monolayer and 3D spheroid culture based on galectin-3 expression and localization

doi: 10.3389/fonc.2022.999158

Figure Lengend Snippet: Migration-related expression in CCA cells in 2D and 3D culture systems. (A) CCA cells were grown as monolayers and spheroids for 6 days. Cells were harvested and lysed. Protein expression was determined by Western blotting. Migration-related proteins were compared between the two culture systems in each cell line. β-actin was used as an internal control. (B) Expressions of galectin-3 was quantified as relative intensity to β-actin. The data represent means and ± standard error. ** p < 0.01, NS represents not significant.

Article Snippet: The galectin-3 shRNA plasmid (sc-155994-SH) containing a pool of three to five lentiviral vector plasmids each encoding galectin-3-specific 19-25 nucleotides (plus hairpin) shRNAs, and scramble shRNA plasmid-A (sc-108060) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Migration, Expressing, Western Blot

Journal: Frontiers in Oncology

Article Title: Distinct cholangiocarcinoma cell migration in 2D monolayer and 3D spheroid culture based on galectin-3 expression and localization

doi: 10.3389/fonc.2022.999158

Figure Lengend Snippet: Localization of galectin-3 in 2D and 3D CCA cells. CCA cells were cultured as monolayer and spheroids for 10 days before harvesting for the immunofluorescence. CCA cells were fixed, permeabilized and blocked before staining with galectin-3 antibody (green). TRITC-phalloidin and Hoechst 33342 were used as counterstains for F-actin (red) and nuclei (blue), respectively. The tumor spheroid images were taken under a confocal microscope using 60x and 120x magnifications. The scale bars in 2D and 3D systems indicate 25 µm and 10 µm, respectively.

Article Snippet: The galectin-3 shRNA plasmid (sc-155994-SH) containing a pool of three to five lentiviral vector plasmids each encoding galectin-3-specific 19-25 nucleotides (plus hairpin) shRNAs, and scramble shRNA plasmid-A (sc-108060) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, Immunofluorescence, Staining, Microscopy

Journal: Frontiers in Oncology

Article Title: Distinct cholangiocarcinoma cell migration in 2D monolayer and 3D spheroid culture based on galectin-3 expression and localization

doi: 10.3389/fonc.2022.999158

Figure Lengend Snippet: Effect of galectin-3 on 2D and 3D KKU-213A cell migration. (A) The relative galectin-3 mRNA level of KKU-213A knockdown cells compared to the control. (B) Representative galectin-3 immunoblots of KKU-213A cells subjected to gal-3 and scrambled shRNA. β-actin was used as the loading control. (C) The relative galectin-3 protein expression of KKU-213A knockdown cells compared to the control. (D) The morphology of galectin-3 knockdown cells in 2D and 3D cultures. The 2D culture images were taken before cells reached confluence and 3D culture images were taken on day 6. (E) Control and galectin-3 knockdown KKU-213A cells were grown as monolayer or tumor spheroids for 4 days. rGal-3 was treated during the migration assay. The migration area of KKU-213A monolayer and tumor spheroids were collected at 0 h and 12 h. The scale bars represent 100 µm. (F, G) Bars represent the relative migration of galectin-3 knockdown cells with and without the treatment of rGal-3 in (F) 2D and (G) 3D conditions compared to the control cells. The data represent means and ± standard error. ** p < 0.01.

Article Snippet: The galectin-3 shRNA plasmid (sc-155994-SH) containing a pool of three to five lentiviral vector plasmids each encoding galectin-3-specific 19-25 nucleotides (plus hairpin) shRNAs, and scramble shRNA plasmid-A (sc-108060) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Migration, Western Blot, shRNA, Expressing

Journal: Frontiers in Oncology

Article Title: Distinct cholangiocarcinoma cell migration in 2D monolayer and 3D spheroid culture based on galectin-3 expression and localization

doi: 10.3389/fonc.2022.999158

Figure Lengend Snippet: Kaplan-Meier survival curve of CCA patients according to galectin-3 expression. The cumulative (A) overall survival ( P = 0.54) and (B) disease-free survival ( P = 0.41) plot stratified by the level of galectin-3 expression.

Article Snippet: The galectin-3 shRNA plasmid (sc-155994-SH) containing a pool of three to five lentiviral vector plasmids each encoding galectin-3-specific 19-25 nucleotides (plus hairpin) shRNAs, and scramble shRNA plasmid-A (sc-108060) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing