Fig 1: Genetic depletion of IKKα increases the metastatic potential of patient-derived organoid cells.A, B Schematic representation of the strategy used for the vivo intrasplenic metastasis assay with PDO5 in nude mice and photograph of the liver metastases (A); and bar chart with the quantification of the number of macroscopic metastases in the different experimental conditions WT, IKKα KO and WT followed by vemurafenib treatment (B) (n = 8 (WT), 7 (KO) and 8 (WT+Vemur.) mice examined). C Representative microscopic images of the immunohistochemistry analysis of the activated fibroblast marker SMA and the proliferation marker KI67 in the livers of mice transplanted with PDO5 of the indicated genotypes, and quantification of the data (n = 6 (WT) and 8 (KO) tumor samples examined). D, E Kaplan-Meier curves of disease-free (DFS) and overall survival (OS) over time for CRC patients included in the metacohort according to CHUK levels using the optimal values as cutoff for patient stratification. Patients at stages II + III (D) or all stages (E) were included in the analysis. Kaplan-Meier curve estimates are shown with a 95% confidence interval. Statistical tests; B Data are presented as mean ± SD, ordinary one-way ANOVA and Tukey’s multiple comparisons test (n.s. p-value = 0.7671); C Data are presented as box plots showing the median, the 25th–75th percentiles, and the minimum and maximum values, unpaired two-tailed t test (n.s. p-value > 0.05). Source data are provided as a Source Data file.
Fig 2: Suppression of IKK function increases TFEB protein levels and enhances lysosomal biogenesis and function.a Hierarchical clustering of all tested drugs based on TFEBNTI distribution curves. The red arrow indicates a cluster of drugs targeting mTORC1, while the green arrow indicates a cluster of drugs affecting TFEB subcellular distribution, but distinct from the mTORC1 cluster. Vehicle controls (DMSO) are indicated in the clustering. b Dot plot displaying TFEB-GFP intensity levels and the product of the distances from DMSO and Torin1 in the hierarchical clustering. TFEB-GFP intensity levels were measured after 6-hour incubation with a drug concentration of 10 µM. c Immunoblot analysis of protein extracts from WT MEFs and MEF lines bearing targeted deletion of the genes encoding IKKα, IKKβ, or IKKγ. IκBα is used as an indicator of IKK activity (n = 3 independent experiments). d Quantification of the immunoblots in (c), normalized to the corresponding GAPDH levels. p-values are calculated based on a two-sided student’s t test. e Relative Tcfeb mRNA levels determined by RT-qPCR in samples shown in panel (c). Results were normalized to Ppia. One-way ANOVA with Dunnett’s multiple comparisons test. f Representative images of immunostaining for LAMP1 (green) and DAPI (blue). Scale bar = 20 µm. g, h quantification of LAMP1-positive area (g) and LAMP1 integrated intensity (h). for WT and IKKα -/- MEFs, n = 32 cells; for IKKβ-/- MEFs, n = 33 cells; for IKKγ-/- MEFs, n = 35 cells. Welch’s one-way ANOVA with Dunnett’s T3 multiple comparisons test. i Representative images of DQ BSA fluorescence (green) and DAPI (blue). Scale bar = 20 µm. j Quantification of the DQ BSA integrated intensity. n = 30 cells each condition. Welch’s one-way ANOVA with Dunnett’s T3 multiple comparisons test. k–m HEK293T cells were transfected with scramble siRNA or siRNAs targeting CHUK (IKKα) (k), IKBKB (IKKβ) (l), or IKBKG (IKKγ) (m). After 48 h, transcriptional analysis was performed, and gene expression levels were normalized to the housekeeping gene, GAPDH (n = 3 independent experiments). Multiple two-sided t tests with Benjamini–Hochberg FDR correction. Results are presented as mean ± SEM. Source data are provided as a Source Data file.
Supplier Page from Abcam for Recombinant human IKK alpha protein (Active)