Fig 2: KCTD15 silencing in SKBR3 causes the inactivation of the NF-?B pathway. (A) Western blot analysis of KCTD15 expression in SKBR3ctrl and SKBR3KCTD15- obtained by CRISP/CAS9 technology. B-Actin was used as the internal control. Numbers represent molecular weight protein markers. (B) Bar-plot diagram of NF-?B proteins pathway in SKBR3ctrl (black bars) and SKBR3KCTD15- (red bars) reported as Mean Fluorescence Intensity of two independent experiments determined by cytofluorimeter. (C) Immunofluorescence experiments of untreated SKBR3ctrl (upper panels), JSH-23 treated (middle panels) and SKBR3KCTD15- (lower panels). Fluorescence microscopy experiments. Column (I) Nuclei staining with DAPI (blue) and FITC- NF-?B RelA (green). Column (II) Nuclei staining with DAPI (blue) and PE-B-tubulin (red). (III) Overlapping of FITC, PE, and DAPI channels. Magnification × 63. Scale bars 20 µm.

Fig 3: CRISPR/CAS9 KCTD15 silencing reduces SKBR3 growth. (A) Cell proliferation at 24, 48 and 72 h of SKBR3 control (black line) and SKBR3KCTD15- (red line). Cell growth was expressed as number of cells/µL. Error bars represent the SD of three independent experiments. * = p-value < 0.05 (B) MTT assay of SKBR3ctrl (Black bar) and SKBR3KCTD15- (red bar) after 72 h of growth. * = p-value < 0.05, ** = p-value < 0.01, Mann–Whitney t-test. Error bars represent the SD of six independent experiments. (C) Cyclins mRNA expression levels in SKBR3ctrl (black bar) and SKBR3KCTD15- (red bar). The relative expression was determined using the 2-?Ct method. Cyclins relative expression is shown as mean +/- SD of two technical independent experiments. * = p-value < 0.05, Mann–Whitney t-test.

Fig 4: KCTD15 mRNA expression in breast cancer different subtypes and normal tissues (GEPIA). Box plots show median mRNA expression levels of KCTD15 in breast cancer tumors (red plots) and the corresponding normal tissues (gray plot). Axis units are Log2 (TPM + 1). * red = p-value < 0.01. T = tumor tissues. N = normal tissues.

Fig 5: KCTD15 expression in pediatric B-ALL patients. (a) KCTD15 intracellular expression measured by mean fluorescence intensity (MFI) for the twelve patients at diagnosis (blue histograms-Patients coded as PA-#) and at day +33 (green histograms- Patients coded as PA-#-R) after therapy. Forward Scatter vs SideScatter plot on the left represent cellular gating strategy. (b) KCTD15 protein levels were measured by FCM for each patient at diagnosis (blue circles) and after therapy (green circles) at day +33. Protein levels significantly decreased from a median value of 5.66*104 to 3.1*103 MFI (***p = 0.0005, Wilcoxon matched-pairs test). The inset reports the statistical parameters for the two ensembles. (c) Fluorescence microscopy experiments of bone marrow smears from four consecutive B-ALL patients. Endogenous KCTD15 was labeled with FITC-conjugated secondary antibody. Column I) KCTD15-FITC fluorescence (green). Column II) Nuclei staining with DAPI (blue). III) Overlapping of FITC and DAPI channels. IV) Enlarged detail of overlapped channels. PA-# = patient-# at diagnosis. Magnification 63 × . Scale bars 20 µm.