EMBO/SourceData huggingface dataset:
Figure 5A. Representative images of time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (green) and SiR-lysosome-stained LE/Lys (orange) treated with either siControl or co-treated with siVAPA, siVAPB and siMOSPD2. Zoom insets show regions of interest containing mobile LE/Lys as denoted by the white arrow. First row represents ER (TMD-GFP) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and SiR-lysosome stained LE/Lys (orange). Scale bar: 5µm, zoom insets 3µm. Images related to movie EV40 and 47. Images were acquired for 90 frames at 1Hz.B. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange) treated with siControl or with siVAPA, siVAPB and siMOSPD2. Zoom insets show region of interest containing ER (TMD-GFP) (green) and junction analysis of skeletonized ER where number of ER junctions representing the quantified average. Scale bars: 10µm. zoom insets: 3µm.C. MDA quantification of ER of panels (A, B) and fig. EV4B-G normalized to control cells showing average ER movement surrounding mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=30 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. **P<0.01, ****P< 0.0001, ns=not significant D. MDA quantification of SiR-lysosome-stained LE/Lys of (A, B) and fig. EV4B - G normalized to 1 showing total average endosomal movement. Graph represents mean ±s.d. of n = 15 cells per condition from 3 independent experiments. Significance two tailed student t-test. ns= not significant. E. Quantification of number of ER junctions per µm2 from (B) and fig. H - M as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell of n = 15 from 3 independent experiments. Significance two-tailed student t-test. *P<0.05, **P<0.01 ***P<0.001, ****P< 0.0001, ns=not significant.
Figure 1  ASPC-1, MiaPaCa-2, Colo-357, Panc-1, Panc-89 and PancTu-1 cells were grown in the absence or presence of the indicated concentrations of omeprazole, 5-fluorouracile, pantoprazole or gemcitabine, respectively, for 4 days to determine the IC50 values. The IC50 ies and the sigmoidicities are gradually differing between the cell lines. In lower concentrations a slight growth-stimulatory effect (hormesis) was observed.
Figure 2  The cell lines ASPC-1, MiaPaCa-2 , Colo357, Panc-1, Panc89 and PancTu1 were untreated or treated with 5-FU alone or in combination with the indicated concentrations of OMP for 4 days. At lower doses of 5-FU alone (black lines, 0 µg/ml OMP) a growth-stimulatory effect (hormesis) was observed in the cell lines ASPC-1, Panc-1 and PancTu-1. The data points show the means of 8 measurements. The curves are fitted in these cell lines using the Brain-Cousens model (see sub-section 4.11). In MiaPaCa-2 and Panc-89 cells no hormesis occured, these curves were fitted using a three parameter logistic model. In Colo357 cells the curves could not be fitted by any pharmacodynamic model due to large standard errors at these lower concentrations (data points shown). The red, green and blue lines indicate the dose-effect curves of 5-FU when various concentrations of OMP were added (10, 20 and 40 µg/ml, respectively). In ASPC-1 and Panc-1 cells the hormesis of 5-FU was reversed and in PancTu-1 cell it was mitigated by OMP depending on the concentration of 5-FU. In MiaPaCa-2 cells we found an additive interaction of 5-FU with OMP. In Panc-89 cells the interaction was antagonistic.
Figure 3  Acridine Orange was added to living untreated ASPC-1 and MiaPaCa-2 cells and cells treated with 5-FU , OMP or the combination of both for 30 minutes or 24 hours. Microscopical life images were taken at 525 nm (green) and 650 nm (red) to detect changes in the lysosomal pH value (three images per plate, three plates per group). The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann-Whitney-U-test. Significant differences compared to control are marked by *. In ASPC-1 cells, after 30 min of treatment, intralysosomal acidity increased upon treatment with OMP (p:0.0051) and 5-FU+OMP (p<0.0001). After 24 hours, the acidity is increased upon all treatment regimens (5-FU - p:0.0002; OMP - p<0.00001; OMP+5-FU - p:0.037). In MiaPaCa-2 cells the acidity is elevated after 30 min upon 5-FU (p:0.005) and decreased after treatment with OMP (p:0.037), 5-FU (p:0.00026) and 5-FU+OMP (p:0.011) after 24 hours.
Figure 4(A) ASPC-1 cell without treatment (800 fold). (B) ASPC-1 cell undergoing apoptosis upon 160 µg/ml OMP after 24 hours (800 fold). Vacuolisation of the cytoplasma and condensation of the nucleus are visible. (C) Phagophores and autophagosomes in a segment of an ASPC-1 cell treated with omeprazole 80 µg/ml for 24 hours (2800fold enlargement). The phagophores are characterised by a cup-like shape (white arrows). Autophagosomes are closed particles, the number of which is increased in treated cells (black arrows). (D) Early phagophores and autophagosomes are also found in MiaPaCa-2 cells treated with OMP 80 µg/ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex. In contrast to ASPC-1 cells, early signs of apoptosis such as vacuolization, are also present.(E) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa-2 and ASPC-1 cells untreated or treated with 5-FU, OMP or the combination of both for 24 hours with standard errors. Significant differences compared to control are marked by *. In ASPC-1 cells there were significant differences compared to the control in the OMP group (p: 0.03) and the 5-FU+OMP group (p: 0.03). In MiaPaCa-2 cells the 5-FU+OMP group differed significantly from control (p<0.001).
Figure 6  The ASPC-1 cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colours of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. The most obvious changes is that the PC/Cho ratio is significantly lower in the OMP group compared to the control group. Upon 5-FU, the Cho/Acetate ratio decrease is the only significant change. In the 5-FU+OMP group, there are several significant changes, i.e.the FACH2/CH = CH ratio is significantly higher. Moreover, the Cho/Acetate ratio changed upon 5-FU+OMP as in the 5-FU group, but also the citrate/GSH ratio. The cellular biochemical effects involve mainly the fatty acid and phospholipid metabolism pointing to membrane anabolism. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline, Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.
Figure 7  The cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colors of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. Upon OMP, the PC/Cho ratios are significantly lower compared to control. Furthermore the acetate/FACH2 ratio is significantly decreased in the OMP group. The latter also showed a higher CH = CH level, the ratio to FACH2 is, however, decreased. Upon 5-FU and 5-FU+OMP, similar changes could be observed. Furthermore, in contrast to ASPC-1 cells, the Ala/Gln/AMP pathway is also involved. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline. Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.
Figure 8(B) Western blot analysis of LAMP-1, a late endosome marker, which was nearly identically distributed over the first two fractions compared to Cathepsin-D, in the controle group, but ocurred only in the first fraction in the OMP treated group. β-COP as an indicator of the Golgi-complex, is found strongly in the lower fraction in the control group, but very weakly in the OMP treated group. Cathepsin - D, an early endosome marker, which can be found in the upper and middle fraction of the control group, but regarding the OMP group, it was only found in the first one.
Figure 9  The experiment was repeated three times with nearly identical results. The LC3-I fractions can be clearly distinguished above the LC3-II signals. Bafilomycin A1 elevated slightly the LC3-II signal intensity compared to control in both cell lines. There was a marked and dose-dependent increase of the signal strength of both fractions when OMP was used. 5-FU alone did not relevantly influence the LC3-level. The corresponding β-Actin level is shown below the LC3-WB to confirm the correctness of this semiquantitative evaluation.
Figure 10  Quantification of mRNAs in ASPC-1 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. The stars indicate significant differences after 24 hours compared to control. The pro-apoptotic bad-mRNA was upregulated after 5-FU- and 5-FU+OMP-treatment. The antiapoptotic bcl-2 mRNA was downregulated by 5-FU and bcl-XL by 5-FU+OMP. OMP led to downregulation of bad and survivin and to upregulation of the mdr-1mRNA.
Figure 11  mRNA quantification in MiaPaCa-2 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. While the mdr-1 mRNA is not signicantly changed, the vATPase mRNA is upregulated in the 5-FU+OMP group after 24 hours compared to control.
Figure 12  The Bad , Puma and Atg12 proteins were detected in MiaPaCa-2 and ASPC-1 cells. The Bad expression is slightly increased by 5-FU and suppressed by OMP and 5-FU+OMP in ASPC-1, but not in MiaPaCa-2 cells. Puma is induced by OMP in both cell lines indicating involvement of the BH3-only pathway. Atg12 is strongly enhanced by OMP in both cell lines. Abbreviations: K - control, F - 5-FU, O - OMP, FO - 5-FU+OMP.
Figure 1E Representative images of Notch activity indicated by Tg(Tp1:mVenus)( green) and matured MG stained with GS (magenta) at different stages. Scale Bars: 20 μm. F Quantitative plots showing the numbers of Tp1+ (left), GS+ (right) cells in the developing retinas at different stages. We collected retinas from 60 hpf (n = 3 dpf (n = 12), 5 dpf (n = 12), and 7 dpf (n = 12, Mean ± SEM; * p<0.05, ***p<0.001, t-test).
Figure 2A Representative images of Notch activation labeled by Tg(Tp1:mVenus)(green) and mature MGs stained with glutamate synthase (GS, magenta) at 4 dpf in Notch ligands (dla-/-/dlb-/-/dlc-/-/dld-/-/jag1b-/-/jag2b-/-) mutants.B, C Quantitative plots showing the numbers of Notch activation (Tp1+) cells (B) and mature MG (GS+) cells (C) in A at 4 dpf. In total, we collected 11 fish for wild-type fish; 8 fish for jag2b-/- mutants; 15 fish for dla-/- mutants; 19 fish for dld-/- mutants; 19 fish for dlc-/- mutants, 11 fish for dlb-/- mutants, 11 fish for jag1b-/- mutants (mean ± SEM; ***p<0.001, t-test).
Figure 4A Upper: representative images showing that GS+ MGs (magenta) were rarely observed in 5-dpf jag2b-/- mutants expressing H2B-CFP (green) driven by the crx promoter. Bottom: representative images showing that GS+ MGs (magenta) were frequently rescued underneath PRs when jag2b expression (green) was driven by the crx promoter in 5-dpf jag2b-/- mutants. The area of retina is labeled with solid line, and each layer structure is labeled with dashed lines and marked with outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Scale bars: 20μm. The remaining MGs are indicated by white arrowheads, and the rescued MGs are indicated by yellow arrows. B Quantification of GS+ MGs (magenta in A) with (green) or without (gray) CFP expression driven by the crx promoter. Ctrl indicates H2B-CFP expression driven by the crx promoter (n = 16 larvae); rescue indicates jag2b-H2B-CFP expression driven by the crx promoter (n = 21 larvae, mean ± SEM; * p<0.05, ns, no significant difference, pair-wised t-test). C The proportions of jag2b-misexpressing PRs (Green) with underneath MGs (GS+, magenta in A) in 5-dpf jag2b-/- mutants and all MGs in retina. In total, we analyzed retinae of 16 control (Ctrl, jag2b-/- , crx::h2b-ecfp expression) and 21 rescue (jag2b-/-, crx::jag2b expression) larvae (Mean ± SEM; ***p<0.001, t-test).
Figure 5D Representative image showing the GS+, but vsx1- expression in the progeny of Tp1+ Müller-like differentiating MGs at wild-type (jag2b+/+). Layer structure indicated in (A). E The percentage of the progeny of Müller-like Tp1+ differentiating MG precursors (red+) expression GS (gray), vsx1 (green), or both negative (magenta) in wild-type (jag2b+/+) fish (n = 14, Mean ± SEM; ***p<0.001, t-test). F Representative image showing vsx1+, but GS- expression in the progeny of Tp1+ Müller-like differentiating MGs in jag2b-/- mutants. Layer structure indicated in (A). G The percentage of the progeny of Müller-like Tp1+ differentiating MG precursors (red+) expressing GS (gray), vsx1 (green), or both negative (magenta) in jag2b-/- mutants (n = 29, mean ± SEM; ***p<0.001, t-test).
Figure 6A Representative images of Notch activation labeled by Tg(Tp1:mVenus)( green) and mature MGs stained with glutamate synthase (GS, magenta) at 4 dpf in Notch receptors (notch1a-/-/notch1b-/-/notch3-/-) mutants. Scale Bars: 20 μm. B Quantitative plots showing the numbers of cells with active Notch (Tp1+) and mature MG (GS+) cells at 4 dpf. In total, we collected 4 fish for wild-type fish; 7 fish for notch1a-/- mutants; 5 fish for notch1b-/- mutants; 18 fish for notch3-/- mutants (Mean ± SEM; ** p<0.01; ***p<0.001, t-test).C Representative images showing that GS+ MGs (magenta) were rarely observed when H2B-CFP expression (green) was driven by the gfap promoter in 5-dpf jag2b-/- mutants. The area between the upper dashed and solid lines is the outer nuclear layer (ONL). The area in the middle two dash lines is the inner plexiform layer (IPL). INL, inner nuclear layer; GCL, ganglion cell layer. D Representative images showing that GS+ MGs (magenta) were frequently rescued underneath cells with notch3 expression (green) driven by the gfap promoter in 5-dpf jag2b-/- mutants. Layer structure is similar as (C). E The cell number of GS+ MGs (magenta) with or without CFP expression driven by gfap promoter. Ctrl: H2B-CFP expression (green) driven by the gfap promoter (n = 5 fish); rescue: notch1b (n = 13 fish) and notch3 (n = 17 fish) NICD-H2B-CFP expression (green) driven by the gfap promoter (Mean ± SEM; * p<0.05, ***p<0.001, ns, no significant difference, pair-wised t-test). F The proportions of notch1b and notch3 NICD-misexpressing cells (green) with underneath MGs (GS+, magenta) in 5-dpf jag2b-/- mutants, indicating that gfap promoter-driven notch1b (n = 13 fish) or notch3 (n = 17 fish) NICD expression could rescue MG loss in jag2b-/- mutants (Mean ± SEM; ***p<0.001, t-test).
Figure 1Maximum projection (2 z-slices) of HeLa cells immunostained for F-actin (Phalloidin), Tubulin, Pericentrin and DAPI at metaphase and early anaphase showing actin accumulation around centrosomes in early anaphase. Scale bar- 10µm Quantification of F-actin (Phalloidin) intensity around pericentrin positive centrosomes in HeLa cells immunostained as in (A), showing the increase in F-actin around centrosomes at early anaphase. Mean actin metaphase = 1 ± 0.03996, n=92, mean actin anaphase = 1.875 ± 0.08895, n=121, Student t-test, ****p<0.0001;Time-lapse sequence from a representative Hela cell expressing Lifeact-GFP transiting from metaphase to anaphase, showing accumulation of actin in the presumptive centrosomal region, red arrows. Images represent 4z-projection. Scale bar-10µm.Time-lapse sequence of a representative HeLa cell expressing GFP-alpha-tubulin and RFP-Lifeact transiting from metaphase to anaphase showing the changes in actin and microtubules. Images represent 4 z-projection of movies taken every 1min. Bottom - higher magnification view of the centrosomes C1 and C2 from time-lapse above showing a transient increase in actin around the centrosomes in early anaphase. Scale bar = 10µm, 1µm in zoom. T=0 is one frame before anaphase onset. Dotted circle shows centrosome position.Integrated fluorescence intensity of GFP-alpha-tubulin and RFP-Lifeact around the centrosomes for a population of cells exiting mitosis, showing actin accumulation around the centrosomes and a simultaneous decrease in tubulin over time. N=30 centrosomes (15 cells). T=0 is one frame before anaphase onset. Graph shows mean +/- SD, values were normalized to intensity at T=0. Quantification and comparison of actin (RFP-Lifeact) and tubulin (a-tubulin-GFP) fluorescence intensity around the centrosomes at metaphase (-4 to -1min), early anaphase (2-5min) and late anaphase (7-10min) for a population of cells. Mean actin intensity at Metaphase =1.002 ± 0.0239, n=97, Early Anaphase = 1.023 ± 0.03047, n=101, Late anaphase = 0.9703 ± 0.05004, n=98. Tubulin intensity at Metaphase = 1.008 ± 0.03356, n=106, Early anaphase = 0.9748 ± 0.04124, n=116, Late anaphase = 0.9276 ± 0.05382, n=101, One-way ANOVA.
Figure 2Stills from time-lapse of HeLa cells expressing GFP-alpha-tubulin and RFP-lifeact arrested at prometaphase with STLC (t=0) and forced to exit mitosis with Cdk1 inhibition (Ro3306) imaged every 90 seconds. Scale bar = 10µm and for zoom = 4µm. n = 21 cells from 4 independent experiments.Quantification of actin around centrosomes when cells are forced to exit mitosis using Cdk1 inhibition as in (A), showing a similar accumulation of actin as observed in bipolar divisions. Graph shows mean actin accumulation normalized to t=0, errors bars indicate standard deviation.HeLa cells expressing GFP-centrin1 arrested at prometaphase or forced mitotic exit (Ro3306 5') and stained with phalloidin. Scale bar- 5µm and for zoom-2µm.The level of actin around the centrosome in (C) was quantified and normalised relative to metaphase and shows an increase during forced mitotic exit. STLC arrest 1 ± 0.01317, n=210, STLC+Ro-5mins=1.251 ± 0.02441, n=144, Unpaired t-test with Welch correction, ****p<0.0001. Quantification of tubulin intensity around centrosomes during prometaphase arrested cells or cells forced to exit with Cdk1 inhibitor. Tubulin intensity decreses during forced mitotic exit. STLC-DMSO- 1 ± 0.02407, n=180, STLC-RO-3306- 0.6516 ± 0.02337, n=136. P<0.0001, Welch's t test.
Figure 3Maximum projection (2-z slices) view of Hela cells pre-treated with DMSO and 0.2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase. Scale bar-10µm. Quantification of actin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the reduction in actin accumulation around the centrosomes following CK666 treatment. DMSO-metaphase-1±0.4325, n=54, CK666-metaphase-0.936±0.4604, n=43, DMSO-anaphase-1.8±0.9736, n=76, CK666-anaphase-1.087±0.4597, n=73; one-way ANOVA, p<0.0001. Date pooled from 3 independent experiments.Z-projection of HeLa cells expressing GFP-centrin1 pre-treated with DMSO or 0.2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F-actin.Quantification of the level of actin around the centrosome from (C), which shows the reduction in actin accumulation around centrosomes following CK666 pre-treatment. DMSO-STLC= 1 ± 0.02768, n=99, CK666 STLC= 0.776 ± 0.02186, n=87, DMSO-RO-3306= 1.339 ± 0.03048, n=127, CK666-RO-3306= 0.7699 ± 0.02246, n=70, one-way ANOVA, p<0.0001.Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit. DMSO-metaphase-1±0.1968, n=54, DMSO-anaphase-0.867±0.2345, n=78, CK666-metaphase-0.7995±0.1275, n=43, CK666-anaphase-0.7407±0.1859, n=74; one-way ANOVA, p<0.0001. Data pooled from 3 independent experiments. Error bars indicated standard deviation.Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, during monopolar exit, showing the failure to reduce tubulin density around centrosomes following CK666 treatment. Data from three independent experiments. Prometaphase arrest (p.a.) 1±0.34 , n=27; forced mitotic exit (f.m.e) 0.78±0.30, n=26; p.a. plus CK666 0.49±0.34, n=30 ; f.m.e plus CK666 0.92±0.37, n=30. One-way ANOVA with Sidak's multiple comparisons test.