Fig 2: Effect of hemin on the proliferation in Caco2 cells. Caco2 cells were treated with hemin (100 nM, 1 µM, and 10 µM) for 48 h. (A) Cell viability of hemin-treated Caco2 cells was measured using the trypan blue exclusion assay (n = 6). (B) LDH released from apoptotic cells in cell-conditioned media was detected by LDH assay (n = 9). (C) Cell number of hemin-treated Caco2 cells was analyzed by trypan blue exclusion assay (n = 7; p = 0.013: Control vs. Hemin 1 µM; p = 0.005: Control vs. Hemin 10 µM). (D) WST-1 levels were measured by a WST-1 cell proliferation assay (n = 9; p = 0.002: Control vs. Hemin 1 µM; p < 0.001: Control vs. Hemin 10 µM). (E) CDK2, CDK4, cyclin D, cyclin E, and ß-actin expression levels were measured by western blot analysis (n = 4; p = 0.015: Control vs. Hemin 1 µM CDK2; p < 0.001: Control vs. Hemin 10 µM CDK2; p = 0.019: Control vs. Hemin 1 µM CDK4; p < 0.008: Control vs. Hemin 10 µM CDK4; p = 0.027: Control vs. Hemin 1 µM cyclin D1; p = 0.026: Control vs. Hemin 10 µM cyclin D1; p = 0.039: Control vs. Hemin 1 µM cyclin E; p = 0.011; Control vs. Hemin 10 µM cyclin E). Quantitative data are presented as the mean ± SE of the mean with scatter plots.LDH, lactate dehydrogenase; WST, water-soluble tetrazolium salt.*p < 0.05.

Fig 3: Cyclin D1, CDK4 and p16 expression in invasive ductal breast carcinomas analyzed by immunohistochemistry . (B) High nuclear cyclin D1 expression. (D) High nuclear CDK4 expression. (F) High p16 expression. (A), (C) and (E) are negative controls for (B), (D) and (F), respectively.

Fig 4: Sequestration of palbociclib into tumor cell lysosomes mediates resistance to chemical CDK4/6 inhibition.(A) Fraction of bromodeoxyuridine (BrdU)–positive cells treated with palbociclib (PALBO) (1 µM) or dimethyl sulfoxide (DMSO) for 24 hours (means ± SD, n = 3). (B) Fraction of BrdU-positive cells transfected with anti-CDK4/CDK6 or control siRNA for 48 hours (means ± SD, n = 3; HCC1954, n = 2). (C and D) Microscopic analysis of HCC1806 cells treated with palbociclib (1 µM) or DMSO for 24 hours and stained with LysoTracker Green (LTR-green) (C), or treated with palbociclib or palbo/bafilomycin A1 (BAF) (100 nM) for 24 hours (D). PALBO auto., palbociclib autofluorescence. Scale bars, 20 µm. (E) Fraction of BrdU-positive cells treated with palbociclib (1 µM) and/or bafilomycin A1 (10nM-SUM149, 25nM-HCC1806/SUM149, 50nM-CAL120) or DMSO for 24 hours (means ± SD, n = 3, two-sided t test). (F) TNBC cells transfected with anti-ATP6AP1 or control siRNAs for 36 hours, stained with LysoSensor Green, and analyzed by fluorescence-activated cell sorting (FACS). (G) BrdU-positive fraction of ATP6AP1-depleted and control cells treated with palbociclib (1 µM) or DMSO for 24 hours (means ± SD, n = 3, two-sided t test). (H) Fraction of BrdU-positive cells treated with palbociclib (1 µM) and/or NH4Cl (50 mM) or DMSO for 24 hours (means ± SD, n = 3, two-sided t test). (I) Fraction of BrdU-positive cells treated with palbociclib, ribociclib (RIBO), abemaciclib (ABEMA) (1 µM), and/or bafilomycin A1 (25 nM) for 24 hours (means ± SD, n = 3, two-sided t test). (J) Fraction of BrdU-positive cells in nontargeting single-guide RNA (snt) or RB1-deleted cells (?RB1) treated with palbociclib (1 µM) and/or bafilomycin A1 (25 nM) for 24 hours (means ± SD, n = 3, two-sided t test).

Fig 5: CDK4 inhibition attenuates the expression of a broad range of p53-responsive genes.a SJSA cells were treated with DMSO, Nutlin, PD0332991 and its combination as indicated in the schedule, followed by RNA deep sequencing analysis. b Heatmap depicting differentially regulated genes sorted according to the z-scores after performing DeSEQ for the condition of Nutlin vs DMSO (n = 2368). Only genes with base mean >20, log2 fold >1, log2 fold <-1 and adjusted p value < 0.05 were taken into consideration. Six samples with biological triplicate are represented. N refers to Nutlin, PD refers to PD0332991 and DMSO as control. RNA-sequencing data of this study were submitted to the GEO, GSE113369. c Gene Set Enrichment Analysis (GSEA) from C2 curated gene sets, provided by the Molecular Signatures Database (MSigDB) v5.052, was performed using variance stabilized RNA-Seq reads from Nutlin (represented as N) and Nutlin+PD0332991 (represented as N+PD30) treated samples. The table was generated by selecting false discovery rate (FDR) < 25% and Enrichment Score (ES) in the descending order. The complete list of pathways is provided in Supplementary Table 2. d Selected enrichment plots from gene sets induced by Nutlin vs Nutlin+PD0332991 are provided as examples