Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2 (2022)
Figure 5

Figure 5 - RNF145 depletion attenuates PA-induced cytotoxicity and promotes membrane fluidity

(A) Loss of ADIPOR2 sensitises cells to PA-induced cytotoxicity. HEK-293T cells were depleted of RNF145 (purple), ADIPOR2 (blue), B2M (CTR; grey), or both ADIPOR2 and RNF145 (blue + purple), exposed to PA (200 µM, 96h) and stained with crystal violet. Quantification of crystal-violet stained cells is shown alongside a representative micrograph (below). The response of each cell line to PA was normalised to its BSA-treated condition (dotted line). Statistically significant differences to cells containing gCTR cells are indicated (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction). Mean ± SD are shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. N = 3 biological replicates, ** q ≤ 0.01. Double knockdowns were generated by sequential constitutive CRISPR/Cas9-mediated depletion of RNF145 or ADIPOR2. Knockdown efficiencies are shown in Appendix Fig S1A.

(B) Cells were treated as in (A) and lysates analysed by immunoblotting. LE, long exposure; *non-specific bands.

(C) HEK-293T cells depleted of B2M (gCTR) or ADIPOR2 (gADIPOR2) were complemented with empty vector (blue), full-length ADIPOR2-HA (green), or a catalytically inactive variant of ADIPOR2-HA (dead ADIPOR2; red) and treated with PA (200 µM, 96h) or BSA (vehicle control, 96h). Measurements from PA-treated cells were normalised to their BSA-treated condition (dotted line). Cell growth was quantified, and representative crystal violet staining is shown. A steady-state anti-HA immunoblot of cell lines used is shown in Appendix Fig S1B. N = 3-5 biological replicates. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. ** q ≤ 0.01 (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction).

(D) ADIPOR2's hydrolytic activity is necessary to prevent excessive accumulation of saturated lipids. HEK-293T cells depleted of ADIPOR2 (gADIPOR2; blue) or B2M (gCTR) were stably complemented with an empty vector control, full length ADIPOR2-HA (green) or catalytically inactive ADIPOR2-HA (red) and incubated with deuterated PA (D3-PA) for 6h. Deuterated lipids derived from internalised D3-PA were quantified by mass spectrometry. The indicated lipids were normalised to the gCTR condition. N = 6 biological replicates. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. * q ≤ 0.05 (one-way ANOVA with post-hoc Benjamini-Hochberg multiple testing correction).

(E) Lipidomic analysis of phosphatidylcholines in HEK-293T cells depleted of RNF145 (gRNF145; white), ADIPOR2 (gADIPOR2; grey), or B2M (gCTR) and subjected to PA treatment (150 µM, 20h). Chain length and saturation/desaturation are indicated. Relative lipid abundance was obtained by normalising to absolute lipid abundance in gCTR cells. Statistically significant differences to gCTR are shown (Wilcoxon Rank Sum Test with Benjamini-Hochberg multiple testing correction). Mean ± SD are shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. N = 6 biological replicates, * q ≤ 0.05.