Journal: Cell Stress & Chaperones

Article Title: Heat shock-induced PI(4)P increase drives HSPA1A translocation to the plasma membrane in cancer and stressed cells through PI4KIII alpha activation

doi: 10.1016/j.cstres.2025.100130

Figure Lengend Snippet: PI4KIII alpha transcript and protein levels remain stable post-heat shock. (a) qPCR analysis of PI4KIII alpha transcript stability using separate batches of cells from all other experiments, including RNA-seq. Transcript levels were assessed at control (37 °C), 0 h post-heat shock, and 8 h recovery and normalized to housekeeping genes (ACTB and GAPDH). HSPA1A was included as a positive control for heat shock response. Statistical significance was determined using one-way ANOVA (Analysis of Variance) followed by the post-hoc Tukey HSD (Honestly Significant Difference) test. (b) Representative Western blot of total PI4KIII alpha protein levels in cell lysates under control conditions, with 0 h post-heat shock and 8 h recovery. HSPA1A is a positive control, demonstrating heat shock-induced upregulation, while GAPDH verifies equal protein loading (complete blots in ). (c) Quantification of Western blot results from independent experiments showing relative PI4KIII alpha and HSPA1A protein levels normalized to GAPDH. Closed circles represent individual data points. Center lines indicate medians; box limits represent the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range; crosses denote sample means. Statistical significance was determined using one-way ANOVA (Analysis of Variance) followed by the post-hoc Tukey HSD (Honestly Significant Difference) test (N.S, not significant).

Article Snippet: • Anti-GAPDH Monoclonal Antibody (Mouse IgG2b; 60004-1-Ig; dilution 1:1000), Proteintech Group, Inc (Rosemont, IL, USA) Blots were incubated with primary antibodies overnight (∼16 h) at 4 °C with constant rotation.

Techniques: RNA Sequencing, Control, Positive Control, Western Blot

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) Co-immunoprecipitation for CUX1 in K562 cells was followed by mass spectrometry (n=2 biological replicates). The heatmap indicates BAF members ranked by the mean label-free quantification fold enrichment compared to IgG controls. (B) Representative co-immunoprecipitation followed by immunoblot in K562 (n=2 biological replicates). (C) K562 CUX1 and SMARCA4 ChIP-seq overlap (n=2 biological replicates, IDR<0.05). (D) Enriched motifs84 at CUX1 and SMARCA4 co-occupied sites. (E) Overlap of SMARCA4 peaks (n=2 biological replicates, IDR<0.05) in gHPRT and gCUX1 K562 cells. (F) Heatmaps showing overlap between CUX1-dependent or CUX1-independent SMARCA4 sites with CUX1. The values are normalized ChIP-seq reads (RPKM). The direct model represents CUX1 recruitment of SMARCA4. The indirect model represents SMARCA4 sites bound but not recruited by CUX1. Example genome snapshots for each category are shown (G).85 (H) Distance to the nearest transcription start site (TSS) of CUX1-recruited and non-CUX1-recruited SMARCA4 sites and hematopoietic TF occupancy (I).

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Immunoprecipitation, Mass Spectrometry, Quantitative Proteomics, Western Blot, ChIP-sequencing

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) Volcano plot comparing ATAC-seq signal in gCUX1 vs. gHPRT K562 cells (n=2 biological replicates). Significance calculated by csaw.41 Top enriched motifs for the significant down sites are shown. (B) H3K27ac ChIP-seq reads (n=2 biological replicates) at significantly down, up and non-significant ATAC sites. (C) CUX1 and SMARCA4 occupancy at down (n=933) vs. CUX1-independent ATAC sites (n=14,256). ATAC-seq signal from gHPRT and gCUX1 cells for CUX1 and SMARCA4 co-occupied sites (D), CUX1-recruited SMARCA4 sites (E), and SMARCA4 sites bound but not recruited by CUX1 (F). Significance for (B-F) calculated by two-sided Wilcoxon rank-sum test.

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: ChIP-sequencing

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) Overlap of CUX1 and SMARCA4 CUT&RUN peaks in primary human CD34+ HSPCs (n=2 biological replicates, IDR<0.05). (B) Genome-wide correlation of CUX1 and SMARCA4 CUT&RUN signals with histone marks from Roadmap Epigenomics.44 All pairwise correlations have p<0.001. (C) CUX1 and SMARCA4 peaks absolute distance (log2 transformed) to the nearest TSS. The dash line indicates 2 Kb. (D) Top GO terms for TSS-proximal and -distal CUX1/SMARCA4 co-bound sites (Bonferroni corrected p-value<0.05).45,46 (E) Volcano plot of ATAC-seq changes in gCUX1 and gHPRT CD34+ HSPCs (n=2 biological replicates). Significance calculated by csaw.41 Top motifs for the down sites are shown. (F) Normalized ATAC reads at genome-wide CUX1-bound enhancers (n=3,902) and a randomly sampled, size-matched list of enhancers not bound by CUX1 (top). Normalized ATAC reads at CUX1-bound enhancers (n=3,902) comparing the control gHPRT and gCUX1 conditions (bottom). (G) Normalized CUT&RUN reads of CUX1 and SMARCA4 in CD34+ HSPC at down vs. CUX1-independent ATAC sites. Significance for (F) and (G) is by two-sided Wilcoxon rank-sum test.

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Genome Wide, Transformation Assay, Control

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) ATAC-seq accessibility for gHPRT and gCUX1 CD34+ HSPCs at distal 3D chromatin contact points looped to CUX1-bound promoters from published CD34+ HSPC Hi-C data.59 (B) IGV snapshot of CUX1 binding at the promoter of KIT and the reduced accessibility of multiple enhancers looped to the promoter. Enhancer and promotor annotations are from Roadmap Epigenomics.44 (C) Integration of CD34+ HSPC ATAC-seq and RNA-seq (n=2 biological replicates).13 Scatterplot shows the RNA log2FC vs. ATAC-seq log2FC for 406 DEGs (FDR<0.1, |log2FC|>0.75). Enriched GO terms related to HSPC lineage commitment are shown.86 (D) Log2FC of ATAC-seq signal comparing CD34+ HSPC gCUX1 vs. gHPRT cells at the hematopoietic cell-type specific enhancers from the VISION database87 (9,657 myeloid enhancers, 11,653 erythroid enhancers, 15,323 lymphoid enhancers), and 10,000 randomly sampled non-cell type specific enhancers. (E) Performance score of cell fate prediction using the published murine HSPC scRNA-seq.68 From left to right: positive control is the top 2,000 genes with the highest cell-cell variation; negative controls are a randomly sampled gene set (n=1,000) and curated list of mouse TFs (n=1,636);88 CUX1-bound genes from human CD34+ HSPC CUT&RUN (n=6,758); overlap of CUX1-bound and differentially-expressed upon CUX1 knockdown in CD34+ HSPC (n=923). Equivalent gene sets were tested for PU.1 and RUNX1 as benchmarks (n = 336 and 325).69–72 For all gene sets larger than 1,000, 50 bootstraps were performed to sample for 1,000 genes. Logistic regression and deep neural network were used to construct the classifier. Significance for (A), (D), (E) is by two-sided Wilcoxon rank sum test.

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Hi-C, Binding Assay, RNA Sequencing, Positive Control, Knockdown, Construct

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: Key resources table

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Purification, Mass Spectrometry, Software

Journal: Nature Communications

Article Title: Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

doi: 10.1038/s41467-019-08942-3

Figure Lengend Snippet: PTex is a fast method to purify cross-linked RNPs. a In vivo cross-linking of HEK293 cells using UV light at 254 nm wavelength results in covalent bonds between RNA and proteins in direct contact. Cross-linked RNPs are indicated by an orange star. b Schematic of the separation principle of biphasic organic extractions used in PTex. Left panel: Phenol-Toluol (50:50) and neutral pH results in an accumulation of proteins and RNA in the upper aqueous phase (aq) while DNA and lipids are retained at the interphase (inter). Right panel: under acidic phenol and chaotropic conditions, non-cross-linked RNA accumulates in the aqueous phase (aq), non-cross-linked proteins in the lower organic phase (org), and cross-linked RNPs (clRNPs) are enriched at the interphase (inter). c Step-by-step analysis of proteins in 9 intermediary steps of the PTex protocol (3 extractions with 3 phases each). Western blot against HuR (ELAVL1, 35 kDa) demonstrates that UV-cross-linking-stabilised HuR-RNA complexes (upper edge/gel pocket of the blot) are largely enriched after PTex (step 3 interphase). A purified fly protein (Sxl RBD4) served as spike-in as 100% non-cross-linked RBP. d 5′-end radioactive-labelled RNA was subjected to PTex in vitro. e PCR with specific primers against exon 5 of the interleukin 3 (IL3) gene demonstrates efficient removal of genomic DNA after either full HEK293 cells (upper panel) or pre-purified genomic DNA (middle panel) were subjected to PTex. A PCR product derived from linear pUC19 DNA (lower panel) is also removed. f Enrichment of known RBPs by PTex tested by western-blot against PTBP1, FUS, or against non-classical RNA-binding enzymes Eno1 and GAPDH. Note that RNaseA treatment was performed after PTex as it removes partially shifted bands (smear) for some RBPs. g PTex enriches for cross-linked RBPs. RNase treatment before PTex strongly reduces recovery of known RNA-binders (PTBP1, FUS). Non-RBP controls Histone H3 and actin (ACTB) are efficiently depleted by PTex ( c , f , g ). For full gels/blots see Supplementary Figures –

Article Snippet: Primary antibodies targeted the proteins HuR (1:1000, Proteintech, 11910–1-AP), ABCF2 (1:1000, Proteintech, 10226-1-AP), CCT7 (1:1000, Proteintech, 15994-1-AP), FUS (1:1000, abcam, ab124923), GAPDH (1:1000, Proteintech, 10494-1-AP), alpha-enolase (ENO1, 1:1000, Proteintech, 11204-1-AP), PTBP1 (1:1000, abcam, ab133734), PABPC1 (1:1000, Proteintech, 10970-1-AP), ACTB (1:1000, Proteintech, 66009-1-Ig), Histone H3 (1:1000, abcam, ab21054), FLAG-tag (1:5000, Sigma, A8592), or Sxl-RBD4 (DHSB, anti-Sxl hybridome culture supernatant M114, 1:20).

Techniques: In Vivo, Western Blot, Purification, In Vitro, Derivative Assay, RNA Binding Assay