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. 2022 May 10;11:e73913. doi: 10.7554/eLife.73913

Figure 5. Bioactivation of other lipidic alkynylcarbinols by specific SDRs.

(A) Cell viability analysis of wild-type HAP-1, DACR clone A4 and AACR clones treated with AAC (S)–4. (B) List of mutations identified on RDH11 by RNA-seq of individual AACR clones. (C) Schematic representation of RDH11 with, in red, the positions of the mutations identified and, in black, the three amino acids critical for catalysis. TM = single-pass transmembrane domain. (D) Structure of all-trans-retinol, a substrate for RDH11. (E) Analysis by immunoblotting of RDH11 levels in wild-type HAP-1, in DACR clone A4 and in the different AACR clones. (F) Analysis by immunoblotting of RDH11 and HSD17B11 levels in wild-type U2OS or clones inactivated for either HSD17B11, RDH11 or both. (G) Cell viability analysis of wild-type U2OS or U2OS clones inactivated for HSD17B11, RDH11, or both and treated with AADC 12. (H) Structure of prostaglandin E2, a substrate of HPGD. (I) Analysis by immunoblotting of GFP and HPGD levels in WT U2OS or U2OS KO HSD17B11 stably complemented with GFP or HPGD-GFP. (J) Cell viability analysis of U2OS or U2OS inactivated for HSD17B11, stably complemented with either HSD17B11-GFP or HPGD-GFP and treated for 72 h with AllAC (S,Sa)- or (R,Sa)–5.

Figure 5—source data 1. Source data related to Figure 5E.
The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc. The regions used to generate the figure are highlighted by back squares in the jpg file, which also contains at the bottom an overlay with a picture of the membrane to locate the protein ladder positions.
Figure 5—source data 2. Source data related to Figure 5F.
The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired using autoradiographic films. Two different immunoblotting of the same extracts were used for this figure (respectively labeled upper and lower). The regions used to generate the figure are highlighted for each immunoblot by back squares in the jpg files.
Figure 5—source data 3. Source data related to Figure 5I.
The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc. The same extracts were loaded twice (left and right part of the membrane) and the membrane sliced to simultaneously probe GFP and HPGD. The regions used to generate the figure are highlighted by back squares in the jpg file, which also contains overlays of the chemiluminescent signal with a picture of the membrane to locate the protein ladder positions.

Figure 5.

Figure 5—figure supplement 1. RDH11 is mutated or underexpressed in all AACR clones.

Figure 5—figure supplement 1.

(A) List of genes carrying near homozygous non- or mis-sense mutations in each of the seven AACR clones analyzed by RNA-seq. For each gene, the impact on the protein sequence is specified. (B) Graphical representation of the genes identified as mutated in each clone. The only gene mutated in more than four clones is highlighted in red. The dashed lines indicate that RDH11 is not expressed in the AACR#B4 clone and carries two heterozygous mutations, T227K and R108* in clone AACR#A6. (C) Histogram representing the ratio between the normalized levels (RPKM) of RDH11 mRNA in each mutant clone as compared to the wild-type HAP-1.
Figure 5—figure supplement 2. RDH11 bioactivates preferentially (S)-AAC over (S)-DAC.

Figure 5—figure supplement 2.

(A) Analysis by immunoblotting of RDH11 and HSD17B11 levels in wild-type U2OS or U2OS inactivated for HSD17B11 (clone B3) or inactivated for both HSD17B11 and RDH11. SAF-A was used as a loading control. (B) Cell viability analysis of U2OS either wild-type, inactivated for HSD17B11 or for both HSD17B11 and RDH11, as described in (A), treated with AAC (S)–4 for 72 h. (C) Analysis by immunoblotting of RDH11 levels in U2OS either wild-type or inactivated for both HSD17B11 and RDH11 using CRISPR/Cas9 and stably complemented with GFP, HSD17B11-GFP or untagged RDH11 (two different clones). Ku80 and total H2AX were used as loading control. (D) Cell viability analysis of U2OS inactivated for HSD17B11 and RDH11, stably complemented as described in (C) and treated with DAC (S)–3 or AAC (S)–4.
Figure 5—figure supplement 2—source data 1. Source data related to Figure 5—figure supplement 2A.
The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc. Two different immunoblotting of the same extracts were used for this figure (respectively labeled upper and lower). The regions used to generate are highlighted for each immunoblot by back squares in the jpg files, which also contain at the bottom an overlay with a picture of the membrane to locate the protein ladder positions.
Figure 5—figure supplement 2—source data 2. Source data related to Figure 5—figure supplement 2C.
The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc. Two different immunoblotting of the same extracts were used for this figure (respectively labeled upper and lower). The regions used to generate the figure are highlighted for each immunoblot by back squares in the jpg files, which also contain at the bottom an overlay with a picture of the membrane to locate the protein ladder positions.