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. 2023 Feb 4;14:616. doi: 10.1038/s41467-023-36168-x

Fig. 2. Osteoclast formation depends on truncated La, but the function of the La domain is dispensable.

Fig. 2

a Representative immunofluorescence images of La in RAW 264.7 prior to mRANKL addition (Cntl.), 3 days post mRANKL addition and 5 days post mRANKL addition when we routinely observe massive, multinucleated osteoclasts like the one imaged here. (α-La mAb). b Representative tris-glycine Western blot of whole cell lysates taken from murine, RAW 264.7 treated as in a (α-La rAb). mRANKL-treated cells were enriched into mononucleated (Mono.) or multinucleated (Multi.) populations as described in the Methods. (Cyclophilin B (α-Cyclo B) loading control). c qPCR evaluation of SSB in human osteoclast precursors treated with siRNA at day 1 post-RANKL addition. (n = 4) (P = 0.0043). d Representative, phase contrast images of non-targeted and SSB-targeted human osteoclasts, as in c, stained for TRAP. e Quantification of the number of fusion events in formation of syncytia with 3+ nuclei in the experiments like the one in d. Fusion was scored at day 3. (n = 3) (P = 0.0306). f Topological illustrations of LMW La 1-375, “uncleavable” La D371A,D374A and “La*” (=La 1-375 Q20A_Y24A_D33I). g Quantification of the number of fusion events in syncytia with 3+ nuclei in RAW 264.7 cells transfected with empty, La 1-375 or La 1-375 Q20A_Y24A_D33I expression plasmids. (n = 3) (P = 0.0213 and 0.0173, respectively). h Representative fluorescence images of human monocyte-derived osteoclasts transfected with empty, La 1-375 or La D371A,D374A expression plasmids. (Magenta = Phalloidin-Alexa488, Grey = Hoechst). i Quantification of the number of fusion events in syncytia with 3+ nuclei in h. (n = 4) (P = 0.0205 and 0.325, respectively). c, e, g, i Statistical significance was evaluated via one-tailed paired t-tests. * = P < 0.05. ** = P < 0.001. Data are presented as mean values + /- SEM. Source data are provided as a Source Data file.