Extended Data Fig. 9: Functional validation of RibosomeST in spermatids and cell lines.
From: A male germ-cell-specific ribosome controls male fertility
a, Stability of PRM2 (P = 3.39 × 10−4) and DYNC1LI2 (P = 0.863) in RibosomeST-/- and wild-type spermatids by cycloheximide chase analysis. Protein level of each protein was normalized to β-Actin and then to time point = 0 h. n = 3. b, Stability of H1FNT-HA in RibosomeST-/- and wild-type N2a cells by cycloheximide chase analysis with the treatment of MG-132. Protein level was firstly normalized to eGFP and then to time point = 0 h (P = 0.049, n = 3). c, Western blotting showing the relative LC3II/LC3I ratio in RibosomeST-/- and wild-type N2a cells without (left, P = 0.100, n = 3) or with (right, P = 0.002, n = 5) H1FNT transfection. Stability of H1FNT-HA in RibosomeST-/- and wild-type N2a cells by cycloheximide chase analysis with the treatment of chloroquine (CQ). Protein level was normalized to eGFP and then to time point = 0 h (P = 0.818, n = 3). d, Stability of PRM2-HA (R28Q: P = 0.021, RPL39L: P = 0.004), H1FNT-HA (R28Q: P = 0.006, RPL39L: P = 0.001), CCIN-HA (R28Q: P = 0.044, RPL39L: P = 0.021), and DYNC1LI2-HA (R28Q: P = 0.208, RPL39L: P = 0.362) in RibosomeST-/- N2a cell lines overexpressing RPL39, RPL39(R28Q) and RPL39L by cycloheximide chase analysis, respectively. Protein level of each protein was normalized to eGFP and then to time point = 0 h. n = 3. e, Unfolded protein response signalling pathway analysis by western blotting of PERK (P = 0.804), eIF2α (P = 0.619), phosphorylated eIF2α (P = 0.608) and ATF6α (P = 2.07 × 10−4) with β-Tubulin as loading control, qPCR of Xbp1 (Xbp1s: P = 0.790, Xbp1u: P = 0.437, n = 4) splicing with 18S as an internal standard, and relative protein expression levels of HSP90B1 (FDR- adjusted q = 0.002, fold change < 1.5, Student’s t-test with BH FDR correction) and HSPA5 (FDR- adjusted q = 0.019, fold change < 1.5, Student’s t-test with BH FDR correction) according to TMT-based quantification by LC-MS/MS (Supplementary Table 2) in wild-type and RibosomeST-/-spermatids. n = 3. f, Distribution of endogenous proteins MOV10, GAPDH and β-Tubulin in the soluble (S) and pellet (P) fractions by western blotting in RibosomeST-/- and wild-type N2a cells. n = 3. g, Distribution of H1FNT-HA (R28Q: P = 0.020, RPL39L: P = 0.013) and DYNC1LI2-HA (R28Q: P = 0.393, RPL39L: P = 0.465) in the S and P fractions by western blotting and the protein expression levels shown as S/P ratio in RibosomeST-/- N2a cell lines overexpressing RPL39, RPL39(R28Q) and RPL39L, respectively. n = 3. h, SDS-resistance assay for protein OAZ3 synthesized in RibosomeST-/- and wild-type N2a cells. n = 3. i, Proportion of different wild-type and mutated alleles in wild-type N2a cells, 1st round of Cas9-edited N2a cells, monoclonal heterozygous Rpl39+/- N2a cell line derived from 1st round of edit, Rpl39+/- N2a cells subjected to 2nd round of Cas9-edit and selection for 4 days by blasticidin and puromycin, with and without additional 7 days of culture without selection. Those without additional 7 days of culture were also subjected to single cell sorting to establish monoclonal cell lines. n = 3. j, Morphology of RPL39L-IRES-eGFP and doxycycline (Dox)-inducible RPL39-overexpressing RibosomeCore-/Y GC-1 cells with or without Dox treatment for different ranges of time. The number of cells was corrected according the split ratio during passage. Scale bar, 20 μm. k, Absolute quantification of RPL39 and RPL39L levels in ribosomes of GC-1 wild-type cell, and RPL39L-IRES-eGFP-overexpressing RibosomeCore-/Y GC-1 cells with or without Dox-induced expression of RPL39 by PRM. n = 3. For a, b, c, d, e, g, i and k, data are mean ± s.e.m. P values were determined using two-tailed Student’s t-tests. n values represent the number of independent experiments. Gel source data are provided in Supplementary Fig. 1.
