Fig. 1: FKBPL mediates ER turnover through autophagy.

a Ratio in cells with fragmented ER were quantified. b, c Numbers of co-localized RFP-KDEL and LAMP1-GFP (b) puncta or GFP-LC3B (c) puncta were quantified. d Schematic representation of mKeima-SEC61B assessing ER-phagic flux. When pH > 7.0, mKeima-SEC61B excites only at 440 nm, representing green; when 7.0 > pH > 5.5, it excites both at 440 nm and 550 nm, representing orange; when pH < 5.5, excites only at 550 nm, representing as red. The illustration created with BioRender.com is released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en. e, f Hep3B cells were co-transfected with mKeima-SEC61B and pcDNA3 or FKBPL and observed by fluorescence microscope (e). Numbers of 440 nm⁻/550 nm⁺ puncta were quantified. g–i HEK293T cells were co-transfected with doxycycline-inducible ssRFP-EGFP-KDEL and pcDNA3.0 or FKBPL for 14 h and cells were maintained in full media containing 10 μg/ml doxycycline for another 8 h. Cell extracts were analyzed by immunoblotting (g). Signal intensities of cleaved relative to full-length ssRFP-EGFP-KDEL were quantified (h). Signal intensities of full-length ssRFP-EGFP-KDEL relative to TUBA were quantified (i). j, k HeLa/WT or FKBPL KO cells were stained with 1 μM ER-tracker for 30 min and observed by fluorescence microscope (j). Fluorescence intensities were measured by ImageJ (k). l, m HeLa/WT or FKBPL KO cells were fixed, immunostained with anti-KDEL antibody, and observed by confocal microscope (l). The fluorescence intensities were measured by ImageJ (m). n–q Extracts of HeLa/WT or FKBPL KO cells were analyzed by immunoblotting (n). Signal intensities of CKAP4 (o), CANX (p), and GRP78 (q) relative to TUBA were quantified. Scale bars in (e, j, l) are 10 μm. Data represent the mean ± SEM of three independent experiments in (a–c, f, k, m, o, p, q) and four independent experiments in (h and i). Differences were statistically analyzed by two-tailed t test in (b, c, f, and h) and by one-way ANOVA followed by Dunnett’s multiple comparisons test in (a, i, k, m, o, p, and q). Arbitrary units, arb. units.