Figure 5 from ANGPTL4 Suppresses Clear Cell Renal Cell Carcinoma via Inhibition of Lysosomal Acid Lipase
Authors: Jin Zeng , De Umasankar , Tithi Tanzia Islam , Kleberg Jeremy , Nataraj Akhila , Jolley Elena , Carelock Madison E. , Davies Brandon S. , Zhang Weizhou , Kolb Ryan 
Published: August 2024

<p>nANGPTL4 inhibits LAL to suppress ccRCC colony formation. <b>A–C,</b> Total intracellular lipids in the indicated CAKi-1 or 786O cells were stained with BODIPY 493/503 and the median fluorescence intensity (MFI) was determined. <b>A,</b> Representative histogram is presented for BODIPY staining of CAKi WT (gray) and A4KO (red) cells. <b>B</b> and <b>C,</b> Graph depicts the average BODIPY 493/503 MFI of the indicated CAKi-1 (<b>B</b>) and 786O (<b>C</b>) cells ± SD. Welch’s <i>t</i> test was used to determine significance. <b>D,</b> CAKi-1 WT and A4KO cells were incubated with BODIPY FL C12 and analyzed by flow cytometry to measure lipid uptake. Left: representative histogram showing BODIPY FL C12 fluorescent intensity of WT (gray) and A4KO (red) cells. Right: graph depicts the average BODIPY FL C12 MFI ± SD in the indicated 786O cells. Welch’s <i>t</i> test was done to determine significance. <b>E,</b> LAL activity was determined by incubating the indicated cells with lysolive-green and measuring the fluorescence Intensity by flow cytometry. Left: representative histogram depicting intensity of lysolive-green fluorescence in CAKi-1 WT (gray) and A4KO (red) cells. Right: graph depicts the average lysolive-green MFI as a fold change compared to WT ± SD. Welch’s <i>t</i> test was done to determine significance. <b>F,</b> Graph depicts the average lysolive-green MFI as a fold change compared to WT ± SD. Welch’s <i>t</i> test was done to determine significance. <b>G,</b> CAKi-1 A4KO cells were mock transfected or transfected with full length (FL) or N-terminus (N-term) ANGPTl4 and incubated with lysolive-green. Left representative histrogram depicting lysolive-green fluorescence intensity in the indicated cell lines. The peak fluorescence intensity in mock transfected cells is indicated by the black line. Right: Average lysolive-green MFI as a fold changed compared to mock transfected ± SD. One-way ANOVA with Dunnett’s test to correct for multiple comparisons was done to determine significance. <b>H,</b> Graph depicts the average number of colonies/well of the indicated CAKi-1 cells grown in the presence or absence of the LAL inhibitor lalistat 1. One-way ANOVA with Dunnett’s test to correct for multiple comparisons was done to determine significance. <b>I,</b> Graph depicts the average number of colonies/well of the indicated 786O cells grown in the presence or absence of the LAL inhibitor lalistat 1. One-way ANOVA with Dunnett’s test to correct for multiple comparisons was done to determine significance. <b>J,</b> CAKi-1 A4KO cells were transfected with siRNA targeting <i>LIPA</i> (gene that encodes LAL) or nontargeting control siRNA (−). Cells were plated in non-adherent conditions, cultured for 72 hours and the number of colonies was counted. Top: western blot for LAL at the indicated time point post transfection. GAPDH blot is used as a loading control. Bottom: graph depicts the average number of colonies per well ± SD. Welch’s <i>t</i> test was done to determine significance. <b>K,</b> CAKi-1 A4KO cells were transfected with a lentivirus plasmid containing CRISPR-Cas9 and one of the three guide RNAs targeting <i>LIPA</i>. After selection total lysates were collected and a Western blot for LAL and GAPDH, as a loading control, was done. Arrow indicates LAL band. <b>L,</b> The indicated CAKi-1 cells (A4KO LIPA KO cells are g2 cells from <b>K</b>) were cultured in nonadherent conditions for 72 hours and the number of colonies was counted. Graph depicts the number of colonies per well ± SD. One-way ANOVA with Dunnett’s test to correct for multiple comparisons was done to determine significance. <b>M,</b> The indicated CAKi-1 cells were cultured for 72 hours, stained with annexin V-FITC and PI and analyzed by flow cytometry. Graph depicts the % of FITC-positive cells as a percentage of single cells ± SD. One-way ANOVA with Dunnett’s test to correct for multiple comparisons was done to determine significance.</p>