Article Figures & Data
Figures
- FIG 1
Pangenomic loss-of-function screen identified SIK2 as essential for TNBC. (A) Schematic for pangenomic siRNA screen. (B) SUM159 cells were transfected with control or ubiquitin B (UBB) siRNA for 96 h. Bars represent relative viability measured by CTG and range (n = 2). (C) Indicated cell lines were exposed to escalating doses of paclitaxel for 48 h. Points indicate viability as measured by CTG (n = 2; bars indicate the ranges). (D) Z-scores for each siRNA, ranked lowest to highest. siRNAs with Z-scores less than −5 are highlighted. (E) Heat map of viability scores (see Table S1 in the supplemental material) of 30 siRNAs identified in panel D in SUM159, SUM149, HCC1806, and WHIM12 with or without paclitaxel ranked by cell line penetrance. Gray coloring indicates unavailable values. (F, left) SUM159 cells were transfected with two independent SIK2 siRNAs. At 120 h posttransfection, cell viability was assessed by CTG. Bars represent average viability (n ≥ 5; ±standard deviations [SD]). (Middle) As described for the left, except mRNA was collected at 72 h and SIK2 transcript measured by qPCR. Bars represent average values and ranges (n = 2). (Right) Whole-cell lysates (WCLs) from SUM159 transfected with the indicated siRNAs for 72 h were immunoblotted with the indicated antibodies.
- FIG 2
SIK2 is essential for viability in a subset of TNBC. (A) Indicated cell lines were transfected with siCTRL or siSIK2 for 120 h. Bars represent relative viability (n = 3; error bars indicate SD) measured by CTG. Solid bars are siRNA pool 1 and bars with hatch marks are pool 2. (B) Indicated cell lines were transfected with siCTRL or siSIK2 for 96 h and then replated at limiting dilution. (C) SUM159 cells were transfected for 24 h and then plated into soft agar. (Left) Images of stained colonies at 2 weeks after plating. (Right) Bars represent average colony numbers and ranges quantitated manually (n = 2). (D) SUM159 cells were exposed to 1 μM vehicle or ARN-3236 for 6 h. (Top) Whole-cell lysates were immunoblotted for the indicated antibodies. (Bottom) Quantitation of band intensity for each antibody relative to ERK1/2 over 2 independent experiments. (E) Indicated cells were plated in 96-well format and treated with the indicated compounds. Bars represent relative viability as determined by CTG assay (n = 8; ±SD). As defined for panel A, red bars indicate siSIK2 sensitive and green bars indicate siSIK2 resistant. (F) SUM159 cells were plated into soft agar and treated each day for 9 days with 1 μM ARN-3236. (Left) Representative images of stained colonies. (Right) Average number of colonies per condition quantitated manually (n = 4; ±SD).
- FIG 3
SIK2 restrict autophagy. (A to C) Immunoblots of WCLs from cells transfected with the indicated siRNAs for 72 h. (D) Representative confocal sections of SUM159-GFP-LC3 cells transfected with the indicated siRNAs for 72 h and exposed to 50 μM Cq for 4 h. Cells were fixed and stained with DAPI. Scale bars, 20 μm. (E) Cells treated as described for panel D were manually quantitated for GFP-positive puncta on an upright epifluorescence microscope. One hundred cells were quantitated per condition over two independent experiments. P values were calculated by Mann-Whitney test. (F) Representative confocal sections of SUM159 cells treated as described for panel D and stained with anti-p62 antibody and DAPI. Scale bars, 20 μm. (G) SUM159 cells treated as described for panel F were manually quantitated for p62-positive puncta on an upright epifluorescence microscope. One hundred cells were quantitated per condition over two independent experiments. P values were calculated by Mann-Whitney test. (H) SUM159-GFP-LC3 cells were exposed to 50 μM Cq for 9 h plus 1 μM ARN-3236 for 24 h. Representative confocal sections are presented. Scale bars, 20 μm. (I) GFP-LC3 puncta was quantitated in cells treated as described for panel H and treated with ARN-3236 for the indicated times. P values were calculated by Mann-Whitney test. (J) U2OS-GFP-LC3 stably expressing control (CTRL) or SIK2(K49M) cDNA were exposed to 50 μM Cq for 4 h. GFP-LC3 puncta in 25 transfected cells were manually quantitated using images acquired by confocal microscopy. P values were calculated using the two-tailed Mann-Whitney test.
- FIG 4
SIK2 restricts autophagy to support TNBC viability. (A) SUM159-GFP-LC3 cells were transfected with the indicated siRNAs for 72 h and exposed to 50 μM Cq for 16 h. Cells were fixed, stained with DAPI, and visualized for GFP. Representative images were captured by epifluorescence. Scale bars, 20 μm. (B) Cells were transfected with the indicated siRNAs for 72 h. WCLs were immunoblotted with the indicated antibodies. (C) Cell lines were transfected with the indicated siRNAs for 96 h (SUM159). Bars represent average relative viability and ranges (n = 2) measured by CTG. (D) Bright-field images of SUM159 cells transfected as described for panel C. (E) SUM159 cells were infected with lentivirus encoding shCTRL or shSIK2 hairpins for 2 days. (Left) Relative SIK2 mRNA expression was measured by qPCR in SUM159 cells stably expressing the indicated shRNAs. Bars represent average levels of SIK2 mRNA and range (n = 2). (Right) WCLs from cells shown on the left were immunoblotted for the indicated antibodies. (F) Cells shown in panel E were plated into soft agar. Bars represent the average number of colonies quantitated manually (n = 3 ±standard errors of the means [SEM]). The P value was calculated using a two-tailed Student t test. (G) Growth curves of SUM159 cells shown in panel E were injected subcutaneously in NSG mice, and tumor volume was measured at the indicated time points. Points represent average volumes of shCTRL (n = 10) or shSIK2 (n = 10) ± SEM. The P value was calculated with an unpaired, two-tailed Student t test. (H) At the study endpoint, tumors were excised and weighed. (Top) Bars represent mean tumor weights ± SEM. The P value was calculated with an unpaired, two-tailed Student t test and are indicated on the graph. (Bottom) Images of tumors expressing the indicated shRNAs.
Additional Files
Supplemental material
- Supplemental file 1 -
Fig. S1 (Cell viability results) and S2 (SIK2 inhibition and mitotic defects)
PDF, 88K
- Supplemental file 2 -
Table S1 (Z-scores for SUM159 cells for siRNA screen)
XLSX, 909K
- Supplemental file 1 -
