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. Author manuscript; available in PMC: 2021 May 15.
Published in final edited form as: Cancer Res. 2020 Aug 19;80(22):4932–4945. doi: 10.1158/0008-5472.CAN-20-1255

Figure 4.

Figure 4.

HSL regulates the invasive impact of oncogenic KRAS. A, Representative images of iKRAS cells cultured in doxycycline (KRASG12D) or following 72 hour doxycycline withdrawal (WT) and stained for LDs (ORO). Scale bar, 10 μm. B, Quantitation of total lipid droplet area per cell described in (A). >50 cells scored in each of five fields for 3 independent biological replicates. C, Western blot of HSL and Actin in mKPC whole-cell lysates constitutively overexpressing HSL or vector control. D, Quantitation of total lipid droplet area per cell of mKPC cells overexpressing HSL or a vector control. >75 cells scored in each of five fields for 3 independent biological replicates. E, Representative immunofluorescence images from cells described in (D) stained for LDs (ORO). Scale bar, 10 μm. F, Quantitation of iKRAS cells overexpressing GFP or HSL-GFP in the presence of doxycycline (G12D) or following 72 hour doxycycline withdrawal (WT) prior to seeding on a Matrigel-coated filter and allowed to invade for 16 hours. >40 cells scored in each of 3 independent biological replicates. G, Quantitation of mKPC overexpressing HSL or vector control and loaded with 200 μM BSA-conjugated OA or BSA control prior to invasion for 16 hours. >245 cells scored in each of 3 independent biological replicates. H, Quantitation of iKRAS cells cultured in the presence of doxycycline (G12D) or following 72 hour doxycycline withdrawal (WT) prior to seeding on a Matrigel-coated filter and allowed to invade. Cells attached for 2 hours prior to drug treatment, and invaded for 14 hours. 10 μM Atglistatin (ATGLi) and 10 μM CAY 10499 (HSL/MGLi) were used. >220 cells scored in each of 3 independent biological replicates. I, mKPC cells transfected with siRNA targeting HSL and loaded with 200μM BSA-conjugated OA or BSA control prior to seeding on a Matrigel-coated filter and invading for 16 hours. >260 cells scored in each of 3 independent biological replicates. Graphs indicate mean ± SEM and analyzed by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.