EMBO/SourceData huggingface dataset:
f9(a) Cytosolic calcium levels were evaluated in iPSC-derived neurons at basal conditions by measuring the 340/380 nm ratio of Fura-2 acetoxymethyl ester. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62). (b) Caffeine (10 mM) was applied to iPSC-derived neurons, and the peak cytosolic calcium concentration was measured as a change in Fura-2 fluorescence. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, Student's t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62).(c) iPSC-derived neurons were treated with A23187 (0-5 μM) for 4 h and cytotoxicity was evaluated by released LDH activity. LDH release values were calculated as the percentage of untreated cells lysed by incubation with Triton X-100. Data are represented as mean+s.e.m.; experiments were independently repeated four times in triplicate. *P0.05, one-way ANOVA, control lines versus mutated lines; δP0.05, one-way ANOVA, mutated lines versus isogenic gene corrected controls.(d) Quantification of TH+ cells treated with A23187 (1 μM) for 4 h expressed as a percentage of DMSO-treated controls. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01, one-way ANOVA.(e) Knockdown efficiency of NECAB2 determined by qRT-PCR and normalized to scrambled non-targeting shRNA. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. ***P0.0001, Student's t-test.(f) Representative western blot for NECAB2 showing knockdown efficiency. 1, scrambled non-targeting shRNA; 2, NECAB2 shRNA.(g) Quantification of TH+ cells in GBA-PD and GD iPSC-derived neuronal cultures infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Values were calculated as a percentage of scrambled shRNA-treated cells. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, Student's t-test.(h) Representative images showing GBA-PD iPSC-derived DA neurons infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Cells were stained for β-TubIII (green) and TH (red). Nuclei were counterstained with Hoechst (blue). (Bar, 20 μm.)
Figure 1(B) Serum CRAMP determination by ELISA (n = 8). Data information: Data in B were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups(E) Localization and expression of CRAMP (red), E-cadherin (green), F4/80 (green) and Ly6G (green) in duodenum by immunofluorescent staining. Representative photomicrographs of individual and merged staining were shown. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Data information: Data were representative from three independent experiments.
Figure 2(C) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..(D) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..(E) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..
Figure 3(D) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.(E) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.(F) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.
Figure 4(A) Western blot and densitometry analyses of gliadin-triggered inflammatory signaling [p-EGFR (Tyr1068), p-Akt, Akt, MyD88, TRAF6, p-p65 and p65] in epithelial cells ex vivo (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.
Figure 5(E-G) The percentages of (E) Foxp3+ Treg, (F) T-bet+ Th1 and (G) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.
Figure 6(D) Duodenal colonizing P. aeruginosa and A. muciniphila as determined by RT-qPCR using bacterial-specific species gene primers (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups(E) The production of LasB (a protease of P. aeruginosa) was examined in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups(H) Duodenal colonizing PA14 and PAO1 as determined by RT-qPCR using P. aeruginosa-specific species gene primer (n = 9). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons
Figure 7(C) The levels of CRAMP in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.(D) Representative images of intestinal damage by H&E staining. Scale bar: 200 μm. The graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.(E) Western blot and densitometry analyses of tight junction proteins (claudin-1 and occludin) and zonulin in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.(I) The percentages of three subsets of CD45+CD11c+ DCs (n = 8). (J-L) The percentages of (J) Foxp3+ Treg, (K) T-bet+ Th1 and (L) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.
Figure 1A. Representative photomicrographs of mouse liver sections following a two-thirds PH of Ki67 positive hepatocytes (brown) visualized by IHC. Images are representative of 3 independent animals per time point.B. Western blot detection of p-MST1, MST1, p-LATS1, LATS1 from proteins isolated from homogenised liver tissue at several time points. TBP was used as a loading control. Results of a single experiment with n = 3 animals per group. Each band is from an independent animal. Protein levels were normalized to TBP and mean value of the ratio of p-MST to total MST and p-LATS to total LATS is plotted.C. Nuclear enriched protein fractions obtained from sham-operated livers and liver 48 h post PH were analyzed by Western blot for YAP expression. Sp1 was used as a loading control. Image is representative of a single experiment with n = 3 animals per group.D. Photomicrograph of YAP1 expression in sham and regenerating liver 48 h post PH visualized by immunoflourescence microscopy. Images are representative of 3 independent animals per condition.E. Livers from sham and 48 h following PH were digested to isolate the hepatocyte population. Protein extracts were analyzed by Western blot for YAP and TAZ expression. β-actin was used as a loading control. Results of a single experiment with n = 2 animals per group. Each band is representative of an independent animal.F. RT-PCR analysis of RNA isolated from mouse liver for Birc5, FoxM1B and cyclin B1. Data are presented as log2 fold change and were calculated using non-operated mouse liver tissue as a control. Unpaired, two-tailed Student t-test was used to determine the significance between the log2 values of livers 6, 24 and 48 h post PH compared to the sham operated controls. Log2 values are plotted on a linear scale as mean ± SD.
Figure 3A. Western blot detection of p-MST1, MST, p-YAP, YAP and TAZ in control and 40 h following PH in young and aged mice. TBP was used as a loading control. Representative results of a single experiment with n = 6 independent animals. Each band is from an independent animal. Western blot quantification ratios are graphically depicted. Proteins levels were normalized to TBP and mean value of the group is plotted. Unpaired, two-tailed Student t-test was used to calculate the significance between 0h (resected liver) and 40h following PH. Error bars represent mean ± SDB. RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, Cyclin A2, Cyclin D1 and p21. RT-PCR data are presented as log2 fold change and each value post PH was calculated for each mouse by comparison to its resected liver lobe. Circle, with red outline marks the aged non-regenerating livers shown in Figure 2B. Each dot is representing an independent animal.
Figure 4A. Eight weeks old mice were injected with liposomes coupled to scrambled siRNA (siScr) or sequences targeting MST1 MST2 (siMST). Each dot is representing an independent animal. The % of Mst1 Mst2 mRNA remaining in the liver was tested by RT-PCR, 1, 3 and 6 days post injection and calculated compared to control non-transfected livers. Unpaired, two-tailed Student t-test was used to calculate the significance of mRNA remaining in comparison to non-transfected control livers at each time point.B. Western blot detection of MST1, p-LATS1 and β-actin 1, 3 and 6 days post siRNA injection. A representative animal from each time point is shown.C. Western blot detection of YAP1, p-YAP1, H3 and p-H3, 3 days post siRNA injection. Two representative animals from each group are shown. All samples were run on the same blot. The blot is split due to non-consecutive loading of the samples.D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, 1, 3 and 6 days post injection. Log2 fold change was calculated using non-transfected mouse liver as control. Unpaired two tailed Student t-test was used to calculate the significance of fold change of in comparison to a panel non-transfected control livers.E. HNF4α and Ki67 were detected by IHC and cells positive for both were classified as proliferating hepatocytes. Ki67 positive hepatocytes are indicated with an asterisk and Ki67 negative bile ducts/biliary cells with an arrow in tissue 3 days post siRNA delivery. The percentages of positive hepatocytes at day 3 and day 6-post injection are graphed. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes in comparison to non-transfected control livers.
Figure 5A. Aged mice (>12 months) were injected i.v. with siScr or siMST. Twenty-four h after siRNA injection a 70% PH was performed and remnant/regenerating liver tissue was harvested 40 h later. p.i. of the livers was calculated by IHC staining of Ki67 positive hepatocytes and based on the percentage of positive cells liver were classified (-) non regenerating or (+) regenerating. Non-surviving animals were sacrificed before the 40 h end time point. "n" indicates the number of animals per group.B. Ki67 immunostaining of the regenerating aged mice livers, 40 h post PH. Sections were scanned using Histotech Pannoramic MIDI Scanner and quantitated with Quant Center 2.0 software. p.i. values are provided on the image.C. Representative photomicrograph of an aged liver treated with siMST and stained with H&amp;amp;E and by IHC for CK19 and Ki67. Arrows are indicating ductal regions with no signs of reaction or oval cell expansion.D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1 at 0 and 40 h post PH. Log2 fold change was calculated using non-transfected/non-resected mouse liver as a control. Representative results of a single experiment with n = 6 animals per group are shown. Paired, two-tailed Student t-test was used to calculate the significant change in signals between liver tissues at the time of resection (0h) compared to 40 h post PH.E. Percentage of Ki67 positive hepatocytes in aged and aged + siMST 40 hours post resection. Representative results of a single experiment with n = 6 independent aged animals for group are shown. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes of siMST treated animals compared to non-treated controls.F. Endpoint liver weight was taken of the remnant lobe and expressed as a percentage to total body weight. Representative results of 1experiment with n = 6 independent animals for group expect for control group were n = 6 are shown. Estimated liver-to-body weight ratio before PH in mice = 3.85 ± 0.05 (SD). Unpaired, two-tailed Student t-test was used to calculate the significance of each aged group in comparison to the control young group.G. Hepatocyte geometric diameter was calculated (see material and methods). Representative results of 1 experiment with n = 3 independent animals per group. Unpaired, two-tailed Student t-test was used to calculate the significant change in hepatocyte size in liver tissue with and without siMST 40 h post PH.
Figure 4(B) Quantification of SARS-CoV-2 infection. After the siRNA knockdown of each target, viral infection was quantified as mean log2 fold-change (log2FC) of the percentage of SARS-CoV-2+ cells relative to the mean of scrambled non-targeting siRNAs ("SCRAMBLED", green dots; accordingly the mean log2FC value of scrambled non-targeting siRNAs was normalized to zero). Predicted positive targets ("TARGETING", grey dots), predicted negative targets ("NEG", blue dots) and positive controls ("POS", red dots, including ACE2 and TMPRSS2) are all shown. Wilcoxon rank-sum test P value comparing the predicted positive targets to scrambled non-targeting siRNAs is given.(C) Quantification of cell number after each target knockdown, calculated as the mean fraction of DAPI+ cells relative to the scrambled non-targeting siRNAs (i.e. the latter was normalized to 1). Colors of dots and P value are interpreted in the same way as in (B).(D) Representative fluorescence images from the siRNA assay showing SARS-CoV-2 infection (green channel, top row), cell number (blue channel, middle row) and merged (bottom row). Results for scrambled non-targeting siRNA as negative control (left column), knockdown of three predicted top metabolic targets (PIKFYVE, SLC16A10, and PIP5K1C, middle columns), and knockdown of positive control (ACE2, right column) are shown. Scale bar=10 μm.
Figure 5(A) PCA plot of the gene expression profiles for the Vero E6 samples from all experimental groups: control (no virus or remdesivir treatment), virus (SARS-CoV-2-infected), virus+remdesivir (SARS-CoV-2-infected treated by remdesivir), remdesivir (remdesivir treatment alone without virus). There are 3 replicates in each group.(C) PCA plot of the average metabolic flux profile computed using the iMAT algorithm (Shlomi et al. 2008; Methods) representative of each of the experimental groups, the labels are the same as (A).
Figure 1(B) Representative leak-subtracted ClC-3, ClC-4 and ClC-5 whole-cell currents measured with (upper row) or without (lower row) 5 mM ATP added to the pipette solution. Currents were elicited every 10 s by voltage steps from 0 mV to +140 mV, followed by a step to -100 mV to maximize the off-gating currents visible as sharp peaks at the end of the pulses. The recording started shortly (10s - 30s) after obtaining a whole-cell configuration. The first and last recorded currents in a series are depicted in green and red, respectively. NMDG-based solutions were used to characterize ClC-4.(C) Effects of various adenine nucleotides or their washout on the ionic transport of ClC-3, ClC-4 and ClC-5. The current amplitudes were measured at the end of the test pulses from experiments as shown in (B) and normalized to the initial current amplitudes obtained after establishing the whole-cell configuration. Some of the experimental data points have been omitted for clarity. Monoexponential fits to the data are depicted as red lines. Asterisks indicate statistical significances evaluated by two-sample t-test analysis of the residual currents determined at the end of each 10-minute measurement (*, p < 0.05; ***, p < 0.001, n.s., not significant; n= 5-11).(D), Left panel: residual currents determined at the end of 10-minute measurements as shown in (C). (D), Right panel: time constants (τ) of the current rundown obtained by fitting monoexponential functions to individual measurements as depicted in (C), (n= 5-11). Asterisks indicate statistical significances evaluated using two-sample t-test analysis (*, p < 0.05; ***, p < 0.001, n.s., not significant).
Figure 2(B) Voltage dependence of the WT ClC-5 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=7-12). Error bars represent SEM. Red lines depict Boltzmann fits to the data; fit parameters are provided in Appendix Table S1.(C) Voltage dependence of the WT ClC-3 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=4-5, Error bars represent SEM.). Red lines represent Boltzmann fits to the data
Figure 3(A) Representative whole-cell recordings of cells expressing E211G ClC-5 upon voltage pulses to +140 mV from a -60-mV holding potential applied in 10-s intervals starting shortly after obtaining the whole-cell configuration with (left) or without (right) 5 mM ATP in the pipette solution. Leak subtraction was not applied. Asymmetric [Cl-] was used (110 mM internal NaCl substituted by TrisSO4) to achieve a stable negative reversal potential of <-50 mV allowing to recognize recordings with high unspecific leak current contamination.(B) Normalized time course of the steady-state E211G ClC-5 current amplitudes measured with (n=5) or without (n=4) internal ATP. Error bars show SEM. Red line represents a monoexponential fit to the data with a time constant of 108±8 s (SEM); currents measured with ATP-free pipette solution declined to 57.9±2.6% (SEM) of the initial amplitude.
Figure 4(A) Effects of different adenine nucleotides or their washout on the ClC-5 current amplitudes (n=4-7). The data were normalized to the initial current amplitude determined shortly after establishing whole-cell configuration. Some of the data points are omitted for clarity. For comparison, the ADP data set from Figure 1 is also depicted. Red lines indicate monoexponential fits to the averaged data.(B) Averaged residual currents determined after 600-s cell dialysis as shown in (A), and normalized to the current at the start of the measurements (n=4-7). Asterisks indicate statistically significant differences from a two-sample t-test analysis with p < 0.001; n.d. - not defined.Averaged rundown time constants obtained from fits with monoexponential decay functions as shown in (A).
Figure 1A-D Appearance of autophagic structures (marked with GFP-Atg8a) during wound closure in the epidermis of control third instar larvae and after epidermal knockdown of the autophagy pathway components Atg1, Atg5, Atg6, Atg7 or Atg12. All constructs are expressed in the epidermis under the control of the A58-Gal4 driver. (A) Time points from movies of wounded epidermis. The wounds have closed by 2 h in all cases. (B) Higher magnification of the areas marked by magenta boxes at t=120 min. (C) Quantification of the appearance of GFP-Atg8a puncta in the imaged area (10000 µm2) 3 control larvae, shown for each individual larva. (D) Quantification of number of GFP-Atg8a puncta in different genetic conditions measured in an area of 10000 µm2 at the time of wound closure; n=4-10 larvae each genotype, for the detail see Data analysis.
Figure 3Control of epidermal autophagy by TOR signalling. (A) Epidermis of third instar larvae expressing the autophagosome marker GFP-Atg8a together with constructs for up- or down-regulating the autophagy pathway in the epidermis. Healthy epidermis contains few autophagosomes, but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes. (B) Higher magnification of the areas marked by magenta boxes in (A).Control of epidermal autophagy by TOR signalling. (C) Quantification of Atg8a puncta in an area of 10000 µm2, n=6-8 larvae each genotype. We assumed unequal sample size and unequal variances and calculations were performed. Values are presented as box plots. Box plot elements are: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers.
Figure 5A-D Fluorescence loss in photobleaching (FLIP) to test free cytoplasmic GFP motility within the epidermis. A small area (magenta circle; 179 or 1098 µm2) was laser-illuminated for the indicated times (3 or 20 min) in control or Atg1-expressing epidermis also expressing free GFP. (A) Snapshots before bleaching and at 3 points of recovery. (B) Kymographs along the broken line in (A) during recovery. (C, D) Quantification of fluorescence recovery after bleaching, shown separately for 3 and 20 min bleaching protocols.
Figure 8B-D Atg1-induced syncytium formation is abolished when Atg5 function is downregulated (compare to rows 2 and 3 in Fig. 5A, B); same representation as shown in Fig. 5A-D.
Figure 9C, D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy. (C) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493/503 to mark lipid accumulations. (D) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions. A-D, n=7-20 larvae each genotype.