Fig 1: High nSOD1 shows an increase in heat shock proteins. Validation of HSPs. (A–C) RT-PCR of HSPA1A (CTRL n = 8; LOW n = 10; HIGH n = 7), HSPA1B (CTRL n = 7; LOW n = 10; HIGH n = 7) and HSPH1 (CTRL n = 13; LOW n = 9; HIGH n = 7) in PBMCs of CTRL, low-nSOD1 sALS patients and high-nSOD1 sALS patients. Data were analyzed by ANOVA (number of analyzed groups = 3) followed by Bonferroni post-test. * p < 0.05. Levels of HSPA1A and HSPA1B mRNAs are higher in patients with high nSOD1, confirming RNA-seq results, while no significant alterations are observed in HSPH1 mRNA through qPCR. (D,E) WB analysis for evaluating expression of HSP70s (CTRL n = 13; LOW n = 10; HIGH n = 9) and HSPH1 (CTRL n = 12; LOW n = 9; HIGH n = 11) in sALS PBMCs. Data were analyzed by ANOVA (number of analyzed groups = 3) followed by Bonferroni post-test. * p < 0.05. Levels of HSP70 and HSPH1 are higher in patients with high nSOD1 compared to those with low nSOD1. Phosphorylation of HSF1 is increased in high-nSOD1 PBMCs of sALS patients. (F) Representative WB membrane for HSP70s and HSHP1. (G) RT-PCR of HSF1 mRNA (CTRL n = 18; LOW n = 10; HIGH n = 8) in PBMCs of CTRL, low-nSOD1 sALS patients and high nSOD1 sALS patients. (H) WB analysis of HSF1 protein. Their levels do not change. (I) WB analysis for the study of HSF1 phosphorylation at serine 326 (CTRL n = 13; LOW n = 9; HIGH n = 10). Levels of phosphorylated HSF1 at S326 are higher in patients with high nSOD1 compared to those with low nSOD1 and to healthy controls. HSF1 Ps326 was normalized to total HSF1. Data were analyzed by ANOVA (number of analyzed groups = 3) followed by Bonferroni post-test. * p < 0.05. (J) Representative WB membrane for pHSF1 and HSF1.
Fig 2: Western blotting analysis of jejunum mucosa proteins, HSPH1 (A), HSPB1 (B), and HSPA1A (C). Data are mean±s.d., n=8 pigs for each group. *P<0.05 and **P<0.01 before vs after heat stress.
Fig 3: ATF2 binds to the HSPH1 promoter to activate its transcription. (A) Venn diagram displays the genome-wide overlap analysis between RNA-seq and ChIP-seq data. (B) Heat map of the differentially expressed genes in ATF2 knockdown MGC803 cells compared with control cells. (C) The relationship between the expression of ATF2 and HSPH1 was examined by correlation analysis based on TCGA database. (D) ChIP-seq peaks of ATF2 enrichment at the promoter region of HSPH1. (E) The HSPH1 protein was analyzed by Western blot in ATF2 stably overexpression and knockdown cells. (F) ChIP-qPCR was used to detect the binding of ATF2 to the HSPH1 promoter in AGS and MGC803 cells. IgG was applied as a negative control. Data are shown as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: Small HSP and HSP110 families have a tendency to be increased under mitochondrial stress.a RNA-seq analysis of HSPs gene expression log2 fold changes (log2FC) in NDUFA11 KO and NDUFA13 KO compared to WT HEK293T cells (n = 4). Up- and down-regulated genes (q-value < 0.05) are shown in green and pink, respectively. The intensity of the color shades depends on the level of expression change. Gray indicates genes with not statistically significant expression changes. b mRNA expression patterns of selected transcripts validated by RT-qPCR. The mRNA levels are presented as fold changes relative to WT. Data shown are mean ± SD (n = 3 biological replicates with two technical replicates). p-value from an ordinary one-way ANOVA with Dunnett’s multiple comparisons test using GraphPad Prism. c Western blot analysis of HSPs expression performed in whole cell lysates of NDUFA11 KO, NDUFA13 KO and WT HEK293T cells. ACTB was used as a loading control. Data shown are representative of three independent experiments. d Quantification of HSPs in western blot analysis normalized to ACTB using ImageJ. The protein levels are presented as fold changes relative to WT. Data shown are mean ± SD (n = 3). p-value from two-sided, unpaired t-test using GraphPad Prism. Source data are provided as a Source Data file.
Fig 5: HSPH1 interacts with SLC7A11 and increases its stability in GC. (A) The interaction between HSPH1 and SLC7A11 was displayed using GeneMANIA analysis. (B) Western blot analysis of the interaction between HSPH1 and SLC7A11 by co-immunoprecipitation. (C–D) The expression of SLC7A11 was analyzed by Western blot and qRT-PCR in ATF2 overexpression AGS cells with or without HSPH1 knockdown by siRNA transfection. (E) ATF2 overexpression AGS cells with or without HSPH1 knockdown were treated with 10 µM sorafenib and 20 µg/ml cycloheximide (CHX) for indicated time points, and then SLC7A11 protein level was analyzed by Western blot. (F–G) Lipid peroxidation and intracellular ROS levels were detected using C11-BODIPY and DCFH-DA staining after sorafenib treatment for 24 h in ATF2 overexpression AGS cells with or without HSPH1 knockdown. (H-I) The cellular MDA and GSH levels were assayed after sorafenib treatment for 24 h in ATF2 overexpression AGS cells with or without HSPH1 knockdown. Data are shown as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.
Supplier Page from Abcam for Anti-Hsp105/HSP110 antibody [EPR4576]