EMBO/SourceData huggingface dataset:
Figure 6(A) Immunoprecipitation of endogenous Bcl‐2 and Beclin‐1. Untransfected HeLa or MV4.11 cells were treated by culture in nutrient‐free EBSS or with ABT737 (1 μM).(B) Immunoprecipitation of wild‐type and ER‐targeted Bcl‐2 with Beclin‐1. Cells stably transfected with wild type, ER‐ and mitochondrion‐targeted Bcl‐2 were subjected to starvation or treated with ABT737, followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1.(C, D) Immunoprecipitation of Bcl‐2 and Beclin‐1 in subcellular fractions. Cells treated as in (B) were lysed and subjected to subcellular fractionation to enrich either ER vesicles (microsomes, (C)) or mitochondria (heavy membrane fraction, (D)), followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1. Calreticulin and Hsp60 were used as internal controls. Results are representative of three independent determinations.
Figure 7(A, B) Interactions between Bcl‐XL, Beclin‐1 and Bad in conditions of autophagy induction. Cells were either treated by nutrient depletion (A) or addition of 1 μM rapamycin, 1 mM lithium chloride, 100 μM L‐690,330 or 50 μM carbamazepine (B), followed by immunprecipitation of Bcl‐XL (as in Figure 2C) and revealing the immunoblots by antibodies specific for Bcl‐XL, Beclin‐1 or Bad.(C) Interaction between endogenous Bad and Bcl‐2. HeLa cells were treated with ABT737 (1 μM) or nutrient‐depleted, followed by immunoprecipitation of Bcl‐2 and immunodetection of Bad.(D, E) Impact of Bad depletion on autophagy. Cells were transfected with LC3‐GFP together with siRNAs specific for Bad, siRNAs specific for emerin or the Beclin‐1‐associated PI3 kinase Vps34. Forty‐eight hours later, when the siRNAs had down‐regulated the proteins of interest (D), cells were subjected by nutrient depletion (NF) or addition of rapamycin (E) and the frequency of cells exhibiting LC3‐GFP aggregation as a marker of autophagy was assessed after 18 h (means±s.d., *P0.05, n=3 separate experiments).(F) Effect of the Bad knock‐out on autophagy. Wild Type (WT) or Bad−/− MEF were transfected with LC3‐GFP and then subjected to nutrient depletion (NF) and/or treatment with ABT737 (18h; means±s.d., n=3). The asterisks denotes a significant (P0.05) effect of Bad deficiency.(G) Effect of the Bad knockout on the Beclin‐1/Bcl‐2 interaction. WT or Bad−/− MEF were subjected to the indicated treatments, followed by immunoprecipitation of Bcl‐2 and immunobloting of Beclin‐1.(H) Overexpression of Bad triggers autophagy. Cells were transfected with LC3‐GFP alone (Control), together with vector‐only or with a vector encoding Bad, and subjected 48 h later to nutrient depletion (NF). Cells were cultured in the continuous presence or absence of the pan‐caspase inhibitor Z‐VAD‐fmk (50 μM) for 16 h, followed by assessment of autophagy as in (E) (means±s.d., n=3 separate experiments, *P0.01). Immunoblots are representative of at least three independent experiments.
Figure 8(A) Representative images of wt and egl‐1 mutant embryos, bearing the plgg−1DsRED(B) Quantification of LGG‐1∷DsRED fluorescence as determined in (A). Each point represents the measurement for one individual embryo. Horizontal bars denote means and gray areas denote standard errors.
Figure 2A Usp27x inhibits the PMA-induced degradation of BimEL in 293FT cells. 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells were treated with dox to induce 3xFlag-Usp27x or Usp27xC87A, or with PMA [16.2 nM] (to induce BimEL degradation), or with the combination of dox, PMA and QVD [10µM] (to inhibit apoptosis) as indicated for 24 h (left) or 48 h (right). Cell lysates were analysed by Western blotting. The blots are representative of n=4 similar experiments. See also Appendix Fig. S5B. * Probably modified form of wild-type 3xFlag-Usp27x (such as dimer, ubiquitinated Usp27x or a stable complex of Usp27x with another interacting partner); this form is only seen if high expression levels of catalytically active Usp27x are reached (see Appendix Fig. S5A where Usp27x was transfected into 293FT cells) or if wt Usp27x is enriched during immunoprecipitation.** Probably degradation product of 3xFlag-Usp27x. These forms of Usp27x were not seen in every experiment.B 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells treated as in A were analysed using conditions of higher resolution. A band very likely corresponding to phosphorylated BimEL is detectable. Similar results were obtained in n=2 separate experiments.C Usp27x stabilizes expression of p-BimEL (Ser69) in Bim-degrading conditions without reducing β-TrCP levels293FT-TetR-3xFlagUsp27x were treated with dox, PMA [16.2 nM], UO126 [10µM] or QVD [10µM] as indicated for 24h, and levels of phosphorylated BimEL at serine69 were analysed by Western blotting using a phospho-Bim (Ser69) specific antibody. Scans are from the same membrane and exposure times. Similar results were obtained in n=2 separate experiments.D UO126 reduces phosphorylation-associated shift of BimEL293FT-TetR-3xFlagUsp27x were treated for 24h with the indicated drug combinations and Bim was detected by Western blotting. UO126 was added 30 minutes prior other stimulation. Similar results were obtained in n=3 separate experiments (see Appendix Fig. S5D).E Usp27x-expression reduces ubiquitination of Bim293FT-TetR-3xFlagUsp27x cells were transfected with a vector coding for 6His-ubiquitin (left) or GFP (right). After 24h cells were treated with PMA and QVD, and 3xFlag-Usp27x was induced by dox at the same time. After additional 20h cells were treated for 4h with MG132 (40 µM) to block proteasomal degradation of ubiquitinated proteins. Cells were lysed under denaturing conditions and His-ubiquitin-labelled proteins or proteins from only GFP expressing cells were purified by Ni2+-NTAaffinity chromatography. Ubiquitinated Bim (Bim-Ubn) was detected by Western blot using anti-Bim antibodies (left). Similar results were obtained in n=3 separate experiments (another experiment including a Coomassie stain is shown in Appendix Fig. S3E). *Indicates non-His-ubiquitinated BimEL that was bound unspecifically to the Ni2+-agarose beads
Figure 3A 293FT-TetR-3xFlag-Usp27x or 3xFlag-Usp27xC87A, a polyclonal derivative where the Bim-locus had been targeted by CRISPR/Cas9- and four single clones obtained by serial dilution from the polyclonal line were treated as indicated (PMA [16.2 nM], QVD [10 µM]). Apoptosis was measured after 24 h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=14 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xC87A, and 3xFlag-Usp27xBim2KO clone #14), n=7 (3xFlag-Usp27xBim2KO clone #11) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown.B The same cells as in A (except Usp27xC87A mutant) were treated as in A and stained for active Bax. Data (means/SEM) are from n=8 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xBim2KO clone #11 and #14) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown. Expression levels of Bim, β-TrCP and Flag-Usp27x of the different cell lines are shown in Appendix Fig. S5E.
Figure 4A Lines based on the human melanoma cell line 1205Lu (BRAF-V600E-positive) were made to carry GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 under the control of a doxycycline (dox)-inducible promoter. GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 were induced with dox for 24 or 48h. Levels of Bim were determined by Western blotting. The two left panels are from the same membrane and exposure times. Similar results were obtained in n=4 separate experiments (left) and n=3 experiments (right). See also Appendix Fig. S6A where 3xFlag-Usp22 is also shown to be unable to increase Bim levels.B A cell line derived from the BRAF-V600E-positive human melanoma line WM1158 was generated to carry inducible GFP-Usp27x. GFP-Usp27x was induced, and Bim was detected as in A. Similar results were obtained in n=3 separate experiments.C Derivatives of the NSCLC cell line HCC827 (expressing constitutively active EGFR) were generated that only express the TetR-repressor or carrying in addition either GFP-Usp27x or GFP-Usp27xC87A. Cells were treated with dox for 72h. Bim was detected by Western blotting. Similar results were obtained in n=2 separate experiments. GFP-Usp27x, GFP-Usp27xC87A, or GFP-Usp22 expression (A-C) was detected using an antibody against GFP.D Caco2 cells carrying a dox inducible HA-BRAF-V600E [37] (left two lanes) or the same cells stably expressing 3xFlag-Usp27x were treated with dox for 48h to induce expression of HA-BRAF-V600E. Bim was detected by Western blotting. The amount of BimEL was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100% (normalized to the tubulin signal for each condition). Similar results were obtained in n=3 separate experiments with varying induction times. See also Appendix Fig. S6D.
Figure 5A Loss of endogenous Usp27x enhances destabilization of BimEL in response to BRAF-V600ECaco2 cells carrying inducible HA-BRAF-V600E were transfected with control siRNA (siCo3) or siRNA directed against Usp27x (combination of three siRNAs targeting Usp27xmRNA) for 24h prior to BRAF-V600E expression with dox for 2h (left) or 4h (right). BimEL was detected by Western blotting. Similar results were also observed in n=2 more experiments after 3h induction (not shown, see Appendix Fig. S6E for siRNA efficacy).B 293FT cells deficient for Usp27x show enhanced destabilization of BimEL in response to PMA293FT wt cells or 293FT-Usp27xKO (clone 2/10, generated using CRISPR-Cas-9) were treated with PMA [16.2 nM] for 16h or 24h to induce BimEL degradation. Western blots show protein levels of n=3 independently performed experiments (similar results were obtained in one more experiment, not shown). See Appendix Fig. S6G for verification of the Usp27xKO cell line by DNA sequence analysis.
Figure 6A 293FT-TetR-3xFlagUsp27x cells were treated with PMA [16.2 nM] plus QVD [10 µM] and 3xFlag-Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide (CHX). Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. Similar results were obtained in n=3 separate experiments. Cut membranes shown were from one membrane with same exposure time. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point.B 1205Lu melanoma cells carrying dox-inducible GFP-Usp27x were treated with dox for 24h. Cycloheximide (CHX, 1µg/ml) was then added. Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. GFP-Usp27x was detected with anti-GFP-antibodies. Similar results were obtained in n=3 separate experiments. A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox-inducible GFP-Usp27xC87A mutant. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point set to 100%. The starting point for the non-Usp27x/C87A inducing cells (-dox) is shown in lane 1 for experiment one and two (-dox, - CHX). Cut membranes shown were from one membrane with same exposure time. Same results were obtained for WM1158 melanoma cells (data not shown).C HCC827 cells were induced to express GFP-Usp27x with dox as indicated. 24h later cycloheximide (CHX, 1µg/ml) was added for the indicated time. BimEL was detected after the indicated times of treatment by Western blotting. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point. Similar results were obtained in n=2 separate experiments. Cut membranes shown were from one membrane with same exposure time.
Figure 7A 1205Lu melanoma cells carrying dox-inducible GFP, GFP-Usp27x or GFP-Usp27xC87A were treated with doxycycline (dox) as indicated. After 48 hUO126 (10µM) was added for another 48 h. Apoptosis was measured by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=3 (GFP) or from n≥6 separate experiments (GFP-Usp27x and GFP-Usp27xC87A). P-values (t-test) for statistically significant differences are shown. N.s., p>0.05. The addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). A stain for active Bax is shown in Appendix Fig. S7B.B HCC827 NSCLC cells carrying dox-inducible GFP-Usp27xC87A, GFP-Usp27x, or two separate lines established from these GFP-Usp27x-cells where the Bim-locus had been targeted for deletion using CRISPR/Cas9 (Bim-KO), were treated with combinations of dox and the EGFR-inhibitor gefitinib as indicated. Apoptosis was measured after 72h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=≥15 (maternal GFP-Usp27x line) or from n≥5 (for Bim1KO) or from n≥6 (for Bim2KO), or n=4 (for GFP-Usp27xC87A) separate experiments. P-values (t-test) for statistically significant differences are shown. Again, the addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). The inset shows Bim-knock-out-efficiency (n=2).
Figure 8Figure 8 Loss of Usp27xreduces gefitinib-induced apoptosis in HCC827 NSCLCHCC827 NSCLC cells and two separate polyclonal lines established from these cells where the Usp27x-locus had been targeted for deletion using CRISPR/Cas9 with two different guide RNA against Usp27x (Usp27xKO-1 and Usp27xKO-4), were treated with gefitinib [10µM] for 48h. Apoptosis was measured by staining for active caspase-3 (A) or by staining for activated Bax (antibody: 6A7 clone) followed by flow cytometric analysis. Data shown in A, B (means/SEM) are from n=4 separate experiments. P-values (t-test) for statistically significant differences are shown.
Figure 1Western blot detection of PAX6 isoforms in human fetal forebrain and retina. The antibody used targets the C terminus of PAX6 and can detect all the 3 isoforms at different molecular weight. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform. #1, #2 are #3 are from ~8-week-old human embryos. #4 is from ~12-week-old human fetus.qPCR quantification of overall PAX6 gene expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). qPCR quantification of PAX6A&B isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). qPCR of PAX6D isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group).Western blot detection of PAX6 isoforms in cells during forebrain and retina differentiation from hESCs. D10 indicates day 10 of differentiation. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform.
Figure 2qPCR quantification of PAX6 expression in Wildtype and PAX6 KO during retinal differentiation (n=3 biological replicates for each group).Western blot detection of PAX6 in Wildtype and PAX6 KO cells at day 20 of retinal differentiation.Immunostaining of PAX6 in Wildtype and PAX6 KO cells. C'-PAX6 antibody detects PAX6A, B and D. N'-PAX6 antibody detects PAX6 A and B but not D. Scale bars: 50 µm.qPCR quantification of retinal related genes in Wildtype and PAX6 KO cells. Samples are collected at day 6, 10, 14 and 19 after the start of differentiation.
Figure 3qPCR verification of PAX6D KO efficiency. PAX6A&B and PAX6D isoforms were also evaluated by qPCR with isoform specific primers (n=3 biological replicates for each group).Western blot detection of PAX6 isoforms in wildtype and PAX6D KO cells.qPCR quantification of retinal gene VSX2 in PAX6D KO cells (n=3 biological replicates for each group).   qPCR quantification of retinal gene SIX6 in PAX6D KO cells (n=3 biological replicates for each group).   Immunostaining of VSX2 in wildtype and PAX6D KO cells. Scale bars:100 µm. Samples were from day 30 differentiating retinal cells.
Figure 4Immunostaining of SOX1 and SOX2 in human fetal forebrain and retinal tissues. Scale bars:50 µm.Flow cytometry analysis of day 7 cell cultures under retinal differentiation from wildtype, PAX6 KO and PAX6D KO cell lines. Flow cytometry analysis of day 20 cell cultures under retinal differentiation from wildtype, PAX6 KO and PAX6D KO cell lines.Quantification of SOX1-/SOX2+ ratio in panel C (n=3 biological replicates for each sample).qPCR quantification of SOX1 in wildtype, PAX6 KO and PAX6D KO cell at day 20 of retinal differentiation (n=3 biological replicates for each sample).
Figure 5qPCR quantification of PAX6A and PAX6D gene expression under the treatment of DOX (n=3 biological replicates for each sample).Western blot analysis of PAX6A and PAX6D protein expression under the treatment of DOX.qPCR quantification of SOX1 gene expression in the PAX6A TetOn and PAX6D TetOn cells (n=3 biological replicates for each group).qPCR quantification of retinal marker expression in PAX6D TetOn cells w/o DOX treatment (n=3 biological replicates for each group).Cell population composition analysis with SOX1 and SOX2 staining from flow cytometry. SOX1/SOX2 double positive cells represent the neuroepithelia fate while the SOX1 negative and SOX2 positive cells represent the neuroretina fate.
Figure 6Verification of PAX6D target genes using PAX6D TetOn cells. Two sets of genes (retinal and non-retinal neural genes) are directly regulated by PAX6D in opposite directions (n=3 biological replicates for each group).
Figure 7qPCR quantification of WNT8B in PAX6D KO cells at multiple time points during retinal differentiation (n=3 biological replicates for each group).qPCR quantification of retinal gene expression under the effect of WNT agonist CHIR99021 and WNT antagonist IWR1 on retinal differentiation (n=3 biological replicates for each group).qPCR quantification of retinal gene expression in PAX6D KO cells treated with WNT antagonist IWR1 to rescue the retinal differentiation (n=3 biological replicates for each group).Western blot of WNT8B and ACTIN in PAX6D KO cells with treatment of untreat, control shRNA lentivirus, WNT8B shRNA lentivirus.qPCR quantification of retinal marker expression in PAX6D KO cells treated with control shRNA lentivirus and WNT8B shRNA lentivirus (n=3 biological replicates for each group).qPCR quantification of retinal gene expression in PAX6D TetOn cells when PAX6D was induced by DOX in the presence of WNT agonist CHIR99021 (n=3 biological replicates for each group).
Figure 1a, TEM analysis of P7 wt retinae (N = 4 eyes) showing a (photoreceptor- Multivesicular Body) PR-MVB in close proximity to (Muller glia) MG (red dashed box). PR-photoreceptor cell, MG (blue dashed lines) -muller glia cell, MVB (yellow and yellow arrow)- multivesicular body, m (green)-mitochondria, cc (pink)-connecting cilium, RPE (purple and purple dashed line) -retina pigment epithelium, RPE melanin-black arrows. Scale bar = 2µm left, 1µm right;f, Representative Dot Blot of 100K EV pellet (each dot represents 3 pooled EV isolations, derived from 60*106 cells; N = 8 experiments) vs cell lysate (CL) from P8 Nrl.Gfp+/+ photoreceptors. EV markers = LAMP1, CD8, CD9; Golgi marker = GM130; phototransduction markers = Recoverin, Rhodopsin, Rod α-Transducin;g, RTqPCR analysis of 100K P8 photoreceptor pellets for Gnat1, Rec, Rho, Crx, Cre and Gfp, relative to β-actin, comparing EVs (N = 8) against the appropriate (Nrl.Gfp+/+ or Nrl.Cre+/-) photoreceptor cell lysate (N = 3). Gnat1, Cre and Gfp mRNA were present in all relevant samples;i, representative flow cytometry analysis of Nrl.Cre+/- & mTmGfloxed co-cultures. Samples were analyzed for expression of myrGFP (recombination) and CD73 (photoreceptor identity) versus CD73-ve fraction (other retinal cells). N > 3 independent cultures for each condition with technical triplicate for each sample; One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test
Figure 2g, Quantification of no. of TdTomato+ve host photoreceptor cells seen following transplantation of live (224 ± 181) versus UV-treated (dead) (3 ± 4) Nrl.Cre+/- P8 photoreceptors (N = 4 eyes per condition). Graph shows mean ± S.D.
Figure 3i & j, Representative images of depth colour coding (ImageJ) of x,y,z live imaging of Nrl.Gfp+/+ photoreceptor cultures showing (i) neurites (blue arrows) growing along the substrate (asterisks denote secondary branching of long GFP+ neurites), and in (j), a free-floating PhNT (magenta arrow). Scale bar = 10 µm;k & l, Quantification of the localization of Nrl.Gfp+/+ processes in the z-axis (depth) (N = 4 independent cultures, n = 1096 cells, nneurites = 440, nnanotubes = 31 where (k) neurites. P values = **** p < 0.0001 (bottom vs top); **p = 0.002 (bottom vs middle); **p = 0.003 (middle vs top) and (l) PhNTs; P-values = **** p < 0.0001 (bottom vs middle & middle vs top) . One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test shows means vary significantly.
Figure 4d, representative time lapse images of cGFP mobility between two PhNT-connected Nrl.Gfp+/+ photoreceptors (dashed ellipse = bleached area) versus an isolated cell (solid ellipse = bleached area) and clustered cells (dot ellipse = bleached area). Images show pre-bleach (t = -5''), bleach (t = 0''), and post-bleach (t = 35'' and 215''). Unbleached reference cells also indicated (arrows); Green = cytoplasm; Scale bar = 5µm.
Figure 5d, Lysosomes can be exchanged, rarely, between PhNT-connected cells. Lysosomes were labelled with SiR-Lyso and their movements were analysed by TrackMate software. Images show deconvolution of 3D reconstructions of lysosomes (surface; red) and cGFP (volume; green) from time-lapse live imaging series of Nrl.Gfp+/+ photoreceptor cultures (green); the position of a transferred lysosome is marked as1 (t = 0), 2 (t = 3'), 3 (t = 6'), 4 (t = 9'); N.B. Deconvolution shows a segment-like process extending from cell "A" and a PhNT connecting cells "A" and "B"; Scale bar = 2 µm;e, Mitochondria can be exchanged, rarely, between PhNT-connected cells. Mitochondria movements determined by TrackMate software. Images show deconvolution of 3D reconstructions of mitochondria (surface; red) and cGFP(volume; green) from time-lapse live imaging series; position of transferred mitochondrion is marked as 1 (t = 0), 2 (t = 6'), 3 (t = 12'), 4 (t = 18'); Mitochondria labelled with mito-Tracker-Orange; Scale bar = 5 µm.