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: Cell Genomics

Article Title: A systems biology approach uncovers novel disease mechanisms in age-related macular degeneration

doi: 10.1016/j.xgen.2023.100302

Figure Lengend Snippet: Pseudo-bulk differential expression from single-nucleus RNA-seq (A–D) Dotplots showing marker gene expression in single-nucleus RNA-seq (sNuc-seq). The color intensity represents the Z score of gene expression, and the dot size represents the percentage of nuclei in each group with non-zero expression. Genes are from pseudo-bulk DE in (A) Müller glia, (B) genes indicating Müller gliosis, (C) RPE, and (D) fibroblasts. (E and F) Examples of pseudo-bulk expression per cell type per donor are in (E) Müller and (F) RPE. (G and H) RNAscope ISH for (G) CRYAB and (H) CLU . Representative images from macular sections of healthy controls (top panels) and GA (middle and bottom panels) from 3 donors each. Nearby sections were stained in (G) and (H), and the images were chosen for close vicinity in each donor. GA images are from the lesion, with lesion centers oriented to the right and the borders to the left. Dashed lines indicate removal of extra white space between the RPE and neural retina that was caused by artifactual postmortem retinal detachment. See also Figure S3 .

Article Snippet: RNAscope probe for human Hs-CRYAB , Advanced Cell Diagnostics , Cat# 426278.

Techniques: Quantitative Proteomics, RNA Sequencing, Marker, Gene Expression, Expressing, RNAscope, Staining

Journal: Cell Genomics

Article Title: A systems biology approach uncovers novel disease mechanisms in age-related macular degeneration

doi: 10.1016/j.xgen.2023.100302

Figure Lengend Snippet:

Article Snippet: RNAscope probe for human Hs-CRYAB , Advanced Cell Diagnostics , Cat# 426278.

Techniques: Recombinant, Protease Inhibitor, RNAscope, DNA Methylation Assay, Sequencing, Methylation, Microscopy, Software

Journal: Melanoma Research

Article Title: Interfering with aggregated α-synuclein in advanced melanoma leads to a major upregulation of MHC class II proteins

doi: 10.1097/CMR.0000000000000982

Figure Lengend Snippet: Proteome and transcriptome changes in human melanoma xenografts treated systemically with anle138b. (a) Comparison of log2FC changes of gene transcript/protein intensities identified by RNA-seq and MS-based proteomics. Candidate transcripts/proteins identified by RNA-seq, MS-based proteomics, or both are colored green, red, and purple, respectively. (b) Correlation of log2FC changes in gene transcript/protein intensity of gene transcripts/proteins selected based upon RNA-seq and MS-based proteomics analysis of WM983-B human melanoma xenografts resected from nude mice that had been treated systemically with anle138b, or had received food pellets not containing anle138b. (c) A tissue section from a nontreated WM983-B melanoma xenograft probed with an antibody to CRYAB. (d) A tissue section from an anle138b-treated WM983-B melanoma xenograft probed with a CRYAB antibody. (e) A tissue section from a nontreated WM983-B melanoma xenograft probed with an antibody to ALDH1A1. (f) A tissue section from an anle138b-treated WM983-B melanoma xenograft probed with an ALDH1A1 antibody. The anti-CRYAB and likewise, the anti-ALDH1A1 antibody-probed tumor xenograft tissue sections (pseudocolored yellow) were counterstained with fluorescent DAPI (pseudocolored blue). (c–f) Scale bar: 50 μm. MS, mass spectrometry.

Article Snippet: Five-μm tissue sections, prepared from cryopreserved WM983-B human melanoma xenografts, were fixed with 4% paraformaldehyde, blocked with 10% goat serum in PBS, and probed with rabbit anti-human antibody against CRYAB (Abcam), ALDH1A1 (Abcam), FN1 (Abcam), or MHC class II (HLA-DPB1) (Abcam).

Techniques: Comparison, RNA Sequencing Assay, Mass Spectrometry

Journal: Stem cell research & therapy

Article Title: Mature human induced pluripotent stem cell-derived cardiomyocytes promote angiogenesis through alpha-B crystallin.

doi: 10.1186/s13287-023-03468-4

Figure Lengend Snippet: Fig. 6 CRYAB was identified as an angiogenic factor of D56-CMs. a Scatter plot of RNA-seq data of D28-CMs and D56-CMs. The red line represents fourfold upregulation in D56-CMs compared with D28-CMs. b qRT-PCR analysis validated the upregulation of CRYAB in D56-CMs. The value for an adult heart sample was set to 1 as a reference. n = 3 per group. c Western blot analysis also confirmed the upregulation of CRYAB protein in D56-CMs compared with D28-CMs. GAPDH protein was used as a loading control. n = 3 per group. d Western blot analysis confirmed the KD of CRYAB (CRYAB-KD) after siRNA treatment in D56-CMs. As a control, non-targeting siRNA was used. n = 3 per group. e CRYAB-KD inhibited HUVEC migration by D56-CMs. The ratio of migrated cells in three independent experiments (n = 3–6 per experiment) is shown. f CRYAB-KD inhibited HUVEC tube lengths by D56-CMs. n = 4 per group. All data are the mean ± SEM, and the P values were determined with unpaired t tests (*P < 0.05, **P < 0.01)

Article Snippet: The Human CRYAB ELISA kit (CUSABIO, Houston, TX, USA; CSB-EL006008HU) was used according to the manufacturer’s protocol.

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Control, Migration

Journal: Stem cell research & therapy

Article Title: Mature human induced pluripotent stem cell-derived cardiomyocytes promote angiogenesis through alpha-B crystallin.

doi: 10.1186/s13287-023-03468-4

Figure Lengend Snippet: Fig. 7 CRYAB-overexpressing D28-CMs enhanced angiogenesis in vivo. a qRT-PCR analysis validated AAV-mediated CRYAB overexpression (CRYAB-OE) 5 days after infection. An AAV vector carrying only tdTomato (tdTomato-OE) was used as a control. n = 4 per group. The value for an adult heart sample was set to 1 as a reference. b Immunostaining also confirmed significant upregulation of CRYAB in CRYAB-OE grafts (left) compared to tdTomato-OE grafts (right). Grafts are indicated by dotted lines. Scale bars, 100 µm. c Representative images of CD31+ microvessel (green) formation in βMHC+ grafts (red) at 4 weeks post-transplantation. Scale bars, 50 μm. d Quantification of microvessel formation in AAV-infected D28-CM grafts. Five sites were randomly selected from each animal. n = 4 per group. All data are the mean ± SEM, and the P values were determined with unpaired t tests (*P < 0.01, ***P < 0.001)

Article Snippet: The Human CRYAB ELISA kit (CUSABIO, Houston, TX, USA; CSB-EL006008HU) was used according to the manufacturer’s protocol.

Techniques: In Vivo, Quantitative RT-PCR, Over Expression, Infection, Plasmid Preparation, Control, Immunostaining, Transplantation Assay