Figure 4. G1/S checkpoint predicts response to combination of palbociclib and autophagy inhibition.

(a) Heat map constructed using the Gene Set Enrichment Analysis program, denoting expression of genes from the Biocarta_Cell_Cycle gene set. (b) Representative TEM microphotograph of parental (Par) and Rb-knockdown MCF7 and T47D cells treated with DMSO or 1 μM palbociclib for 6 days. Scale bars, 500 nm. Rb-knockdown MCF7 and T47D cells were treated with the combination of 1 μM palbociclib and 15 μM HCQ for 6 days, allowed to recover for 6 or 4 days and subjected to (c) Clonogenic assay and (d) Cell counting. MCF7 and T47D cells overexpressing vector (Vec), full-length cyclin E (EL) or LMWE were treated with palbociclib for 6 days and subjected to (e) Dose–response studies to measure Palbociclib IC₅₀ values and (f) Cell counting in combination with 15 μM HCQ. (g,h) Measurement of cellular ROS levels and quantification (MFI; numbers on graph) in Rb-knockdown (g) LMWE overexpressing (h) MCF7 and T47D cells treated palbociclib for 6 days. (i,j) Cell counting (i) and clonogenic assay (k) to assess proliferation upon treatment with 1 μM palbociclib, 1 μM letrozole and 15 μM HCQ in the indicated combinations. (k,l) Impact of overexpressing LMWE in MCF7 aromatase-expressing cells measured by cell counting (k) and clonogenic assay (l) following treatment with 1 μM palbociclib, 1 μM letrozole and 15 μM HCQ in the indicated combinations. (m) IC₅₀ values of letrozole and anastrazole in parental versus letrozole or anastrazole resistant cells, respectively. MCF7 parental, letrazole and anastrazole resistant cells were treated with palbociclib for 6 days and subjected to (n) Dose–response studies to measure Palbociclib IC₅₀ values and (o) Cell counting in combination with 15 μM HCQ. All data represent mean±s.d. from three independent experiments; P values were calculated in comparison to cells treated with DMSO (Control) or parental unless indicated. NS: P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.