Journal: Communications Biology

Article Title: CRYAB_K92 lactylation drives hypertrophy of the ligamentum flavum via an S100A16/RAGE-mediated glycolysis-fibrosis positive feedback loop

doi: 10.1038/s42003-026-10103-4

Figure Lengend Snippet: A The quantitative results of the Pearson’s Correlation Coefficient (PCC). B The Violin plot showing the distribution and differences in the intensity values of modification sites among different samples. C The expression heatmap of differentially modified sites, where red represents high expression, blue represents low expression, and gray represents non-quantifiable values in the corresponding sample. D–G The GO and KEGG pathway enrichment analyses of lactylation-activated proteins are presented via bubble plots (where D: MF, molecular function; E: BP, biological process; F: CC, cellular component; G: pathway enrichment). H Functional annotation of identified proteins. I The Volcano plot of differentially modified proteins and sites between LF/HLF groups. The information of the top five differentially modified sites is also marked in the figure. J The MS/MS spectra including C-terminal y-ions and N-terminal b-ions of CRYAB_K92.

Article Snippet: Recombinant S100A16 (MedChemExpress) was immobilized on a CM5 sensor chip, and serially diluted recombinant CRYAB (MedChemExpress) was injected as the analyte.

Techniques: Modification, Expressing, Functional Assay, Tandem Mass Spectroscopy

Journal: Communications Biology

Article Title: CRYAB_K92 lactylation drives hypertrophy of the ligamentum flavum via an S100A16/RAGE-mediated glycolysis-fibrosis positive feedback loop

doi: 10.1038/s42003-026-10103-4

Figure Lengend Snippet: A The co-IP showed the lactylation level of CRYAB in LF/HLF tissue. B The co-IP showed the regulation of exogenous lactate on the lactylation level of CRYAB in HLF cells. C The co-IP showed the lactylation level of CRYAB in HLF cells before and after the mutation at the CRYAB _K92 site. D , E The western blot and the quantification analysis showed the regulatory effect of exogenous lactate combined with the mutation at the CRYAB _ Kla92 on the fibrosis level in HLF cells. F , G The western blot and the quantification analysis showed the efficiency of P300 silencing and overexpression. H The co-IP showed the regulation of cellular lactylation level of CRYAB by the site mutation in combination with the lactylation-modifying enzyme P300 and the P300 inhibitor C646. I , J The western blot and the quantification analysis showed the regulatory effects on the fibrosis level of HLF cells before and after CRYAB _K92 mutation, as well as in combination with P300 and C646. K–M The results of EDU and FCM assays showing the regulation of the proliferation and cell cycle of HLF cells by the mutation at the CRYAB _K92 site in combination with P300 and C646. Scale bar=50 μm. O The co-IP results compared the regulatory effects of K92 site mutation, P300 knockout, and the use of the P300 inhibitor C646 on CRYAB lactylation in HLF cells. P , Q The western blot and the quantification analysis results compared the regulatory effects of K92 site mutation, P300 knockout, and the use of the P300 inhibitor C646 on the fibrosis level of HLF cells. The quantitative data are presented as mean ± SD, with individual data points overlaid to show the distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Recombinant S100A16 (MedChemExpress) was immobilized on a CM5 sensor chip, and serially diluted recombinant CRYAB (MedChemExpress) was injected as the analyte.

Techniques: Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Over Expression, Knock-Out

Journal: Communications Biology

Article Title: CRYAB_K92 lactylation drives hypertrophy of the ligamentum flavum via an S100A16/RAGE-mediated glycolysis-fibrosis positive feedback loop

doi: 10.1038/s42003-026-10103-4

Figure Lengend Snippet: A The scatter plot shows differentially expressed proteins following mutation of the Kla92 site. S100A16 expression was significantly downregulated after the mutation. B Molecular docking prediction between S100A16 and CRYAB. The binding pose with the lowest PIPER pose energyis displayed. C The sensorgram of Surface plasmon resonance (SPR) analysis confirms a specific and direct interaction between S100A16 and CRYAB. D , E The western blot and quantitative analysis show that the protein half-life of S100A16 was significantly shortened after the CRYAB_K92 mutation. F The qRT-PCR results showed the mutation of the CRYAB _K92 site significantly reduced the mRNA expression level of S100A16 in HLF cells. G Actinomycin D chase analysis of S100A16 mRNA stability in CRYAB _K92 WT and Mut cells. mRNA levels at each time point were normalized to the respective 0-hour value (set as 100%). H , I The cellular immunofluorescence showed that after the CRYAB _K92 mutation, the signal levels of both lactylation (Kla, red) and S100A16 (green) decreased in HLF cells. The merged image shows co-localization between Kla and S100A16 signals. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. J , K The cellular immunofluorescence showed that the mutation of CRYAB _K92 led to reduced expression levels of both CRYAB (red) and S100A16 (green) in HLF cells. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. L The tissue immunofluorescence analysis showed that compared with LF tissue, S100A16 (red) expression was significantly increased in HLF tissue. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. M The co-IP assays using a CRYAB antibody confirmed that the CRYAB _K92 mutation significantly reduced the level of S100A16 protein that interacts with CRYAB. The quantitative data are presented as mean ± SD, with individual data points overlaid to show the distribution. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant S100A16 (MedChemExpress) was immobilized on a CM5 sensor chip, and serially diluted recombinant CRYAB (MedChemExpress) was injected as the analyte.

Techniques: Mutagenesis, Expressing, Binding Assay, SPR Assay, Western Blot, Quantitative RT-PCR, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Communications Biology

Article Title: CRYAB_K92 lactylation drives hypertrophy of the ligamentum flavum via an S100A16/RAGE-mediated glycolysis-fibrosis positive feedback loop

doi: 10.1038/s42003-026-10103-4

Figure Lengend Snippet: A , B The results of western blot analysis showed the regulation of CRYAB_K92 in cooperation with S100A16 on the fibrosis and the RAGE pathway. C–F The results of EDU and FCM indicate that CRYAB_K92 in cooperation with S100A16 can promote the proliferation of HLF cells. Scale bar = 50 μm. G , H The results of western blot showed the regulation of CRYAB _K92 and S100A16 on the fibrosis of HLF cells and the RAGE pathway in combination with NaLA. I The lactic acid content in HLF cells treated with CRYAB _K92 site mutation, exogenous lactic acid, and S100A16 silencing. J The ATP production in HLF cells treated with CRYAB _K92 site mutation, exogenous lactic acid, and S100A16 silencing. K The ECAR in HLF cells treated with CRYAB _K92 site mutation, exogenous lactic acid, and S100A16 silencing. The quantitative data are presented as mean ± SD, with individual data points overlaid to show the distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Recombinant S100A16 (MedChemExpress) was immobilized on a CM5 sensor chip, and serially diluted recombinant CRYAB (MedChemExpress) was injected as the analyte.

Techniques: Western Blot, Mutagenesis

Journal: Communications Biology

Article Title: CRYAB_K92 lactylation drives hypertrophy of the ligamentum flavum via an S100A16/RAGE-mediated glycolysis-fibrosis positive feedback loop

doi: 10.1038/s42003-026-10103-4

Figure Lengend Snippet: The hyperlactate state and lactylation of CRYAB_K92 promote fibrosis and hyperproliferation of ligamentum flavum cells by upregulating S100A16 and activating the RAGE pathway.

Article Snippet: Recombinant S100A16 (MedChemExpress) was immobilized on a CM5 sensor chip, and serially diluted recombinant CRYAB (MedChemExpress) was injected as the analyte.

Techniques:

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the methylation of CRYAB in vitro which exerted anti‐inflammatory effects. (A) BSP was performed to detect CRYAB methylation (white represented unmethylation, black represented methylation). (B) In rat astrocytes, Western blot was applied to check the changes of key factors in DNA methylation (DNMT1, DNMT3a, and DNMT3b) ( n = 3). (C) In rat astrocytes, the positive cells of CRYAB were checked using immunofluorescence. (D) In rat astrocytes, CRYAB protein expression was checked using Western blot ( n = 3). (E) In rat astrocytes, the mRNA expression of Serping1, C3, Sphk1, and S100A10 was checked by RT‐PCR ( n = 3). (F) In rat astrocytes, the protein expression of Serping1, C3, Sphk1, and S100A10 was checked using Western blot ( n = 3). (G) In supernatant of rat astrocytes, the levels of IL‐1β and IL‐6 were examined by ELISA ( n = 3). Data in (B, D, E, F, and G) were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking in 5% non‐fat milk for 1 h, membranes were incubated with primary antibodies against MAFG, C3, CRYAB, Serping1, DNMT3a (Abcam); S100A10, TNF‐α, IL‐1β (Proteintech); Sphk1, IL‐6, DNMT3b (Santa Cruz Biotechnology, USA); β‐actin and DNMT1 (Cell Signaling Technology, USA) overnight at 4°C, followed by incubation with the secondary antibodies (Abcam) for 1 h. To measure the optical density of protein bands, ImageJ software (NIH, USA) was used.

Techniques: Methylation, In Vitro, Western Blot, DNA Methylation Assay, Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the activation of A1 astrocyte and neuroinflammation via CRYAB methylation. (A and B) In rat astrocytes, the mRNA expression of Serping1, C3, Sphk1, and S100A10 was checked by RT‐PCR ( n = 3). (C and D) In rat astrocytes, the protein expression of Serping1, C3, Sphk1, and S100A10 was checked using Western blot ( n = 3). (E and F) In supernatant of rat astrocytes, the levels of IL‐1β and IL‐6 were examined by ELISA ( n = 3). All data were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking in 5% non‐fat milk for 1 h, membranes were incubated with primary antibodies against MAFG, C3, CRYAB, Serping1, DNMT3a (Abcam); S100A10, TNF‐α, IL‐1β (Proteintech); Sphk1, IL‐6, DNMT3b (Santa Cruz Biotechnology, USA); β‐actin and DNMT1 (Cell Signaling Technology, USA) overnight at 4°C, followed by incubation with the secondary antibodies (Abcam) for 1 h. To measure the optical density of protein bands, ImageJ software (NIH, USA) was used.

Techniques: Activation Assay, Methylation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay