EMBO/SourceData huggingface dataset:
Figure 4(A) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2+/+ and LRRK2(G2019S)-expressing MEFs treated with vehicle (Control) or tunicamycin (1 μg/ml). Endogenous PERK, MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Data represent the ratio of phosphorylated to total protein levels. Error bars represent ±SD from independent experiments. Data information: For graph A the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01(B) Immunoprecipitation/immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in MEFs in the of tunicamycin (5 μg/ml). LRRK2(G2019S) MEFs were also pretreated with LRRK2-IN-1 (1 μM). MEFs were transfected with each E3 ubiquitin ligase and biquitin and lysates were immunoprecipitated with antibody against the Myc or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the Myc epitope, phosphoserine or mitofin2 as indicated to the right of each panel. Mfn2: mitofusin 2, Ub: ubiquitin. Data represent ratios of phosphorylated to total E3 ubiquitin ligase and mitofusin2 to actin. Error bars represent ±SD from four independent experiments. Data information: For graph B, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01
Figure 5(A) Peak values of Ca2+ transients in MEFs treated with LRRK2-IN-1 (1 μM). Error bars represent ±SD from six independent experiments. Data information: For graph A , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01(B)(Upper panel) Diagram showing full-length PERK Myc at the N-terminus and FLAG at the C-terminus (M-PERK-F). S1P recognition sequence R33SLL is mutated to A33SLL (M-PERK(R33A)-F). (Lower panel) Immunoblot of PERK and PERK(R33A) from transfected MEFs under tunicamycin. MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method. Lysates were immunoprecipitated with antibody against FLAG. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody as indicated to the right.(C) In vitro kinase assay using isolated PERK, isolated E3 ubiquitin ligase and [γ-32P]ATP. Reaction mixture was subjected to SDS/PAGE. Blots were with antibody against Myc, FLAG or phosphoserine. [γ-32P]ATP-incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , and right blot for 36 ).(D) In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases, HA-tagged Ubl, and His-tagged mitofusin2 in the presence of E1 enzyme, UbcH7 and ATP. E3 ubiquitin ligases were initially phosphorylated PERK or PERK(K618R) in vitro. Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination. itofusin 2 with Ni-NTA subjected to SDS/PAGE. Blots were with antibody against HA or mitofusin2. Mfn2: mitofusin 2, Ub: ubiquitin.(E) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2(G2019S)-expressing MEFs transfected with the increasing amounts of LRRK2-d1-V5 (1, 5, 25 μg/106 cells) treated with tunicamycin (1 μg/ml). Endogenous MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Endogenous mitofusin 2 was immunoblotted with anti-mitofusin 2 antibody. Data represent the ratio of phosphorylated to total protein levels of MARCH5, MULAN and Parkin, and the ratio of mitofusin 2 to actin. Error bars represent ±SD from independent experiments. Data information: For graph E, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01
Figure 6(A) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK and the binding to mutant LRRK2. HEK293 cells were transfected with PERK and either MARCH5, MULAN or Parkin, LRRK2, LRRK2(D1994A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc or V5, and precipitates were immunoblotted with antibody indicated the right.(B) In vitro kinase assay for auto-phosphorylated LRRK2 using isolated LRRK2 mutants and [γ-32P]ATP with or without LRRK2-IN-1 (1 μM). Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS/PAGE. Blots were with antibody against V5 or phosphoserine. [γ-32P]ATP-incorporated LRRK2 visualized by autoradiography.(C) Binding assays to detect interactions between phosphomimetic LRRK2(S1292D) or phosphorylation-defective LRRK2(S1292A) and E3 ubiquitin ligases. HEK293 cells were transfected with LRRK2 mutants and E3 ubiquitin ligases lysates were immunoprecipitated with antibody against Myc or V5 epitope. Precipitated proteins were subjected to SDS/PAGE, for immunoblotwith antibody against V5 or Myc.(D) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK-ΔN in the presence of LRRK2(S1292A) or LRRK2(S1292D). HEK293 cells were transfected with MARCH5, MULAN, Parkin, and PERK-ΔN LRRK2(S1292A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc, and precipitates were immunoblotted with antibody indicated the right.
Figure 7(A) Immunoblot of LC3 and p62. MEFs were transfected with PERK-ΔN or PERK shRNA. Endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph A, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05.(B) Peak values of Ca2+ transients in MEFs transfected with PERK-ΔN or PERK shRNA. Error bars represent ±SD from six independent experiments.
Figure 1Left panel, a scheme of chromosome 18 copy number alterations depicting the distal long arm loss across TCGA Pan-Cancer dataset. Vertical red line indicates the localization of VPS4B. Right panel, enlarged fragment of chromosome 18 showing frequent deletions of VPSB locus in cancer samples. Deletions are marked in blue, amplified regions are marked in red. Analysis of VPS4B copy number alterations in TCGA Pan-Cancer dataset. Cancer types were sorted according to the mean VPS4B copy number after removing germline values.Scatter plot, analysis of VPS4B mRNA expression (number of transcripts per million) plotted against VPS4B copy number from TCGA CRC patient samples (n=376); plot generated using cBioPortal Gao et al, 2013(). Pie chart, summary of all types of VPS4B copy number alterations based on the analysis of data from 615 CRC samples deposited in the Colorectal Adenocarcinoma (TCGA, Provisional)qPCR analysis of VPS4B mRNA abundance in normal colon, adenoma and CRC samples. Adenocarcinoma (n=26); adenoma (n=42); normal colon (n=24). Green horizontal bars indicate means and red whiskers indicate SD.Examples of immunohistochemical staining of VPS4B in normal colon and matched CRC samples as an illustration of the scoring system used for the evaluation presented in (F). 3+ - very intensive staining, 2+ - medium-intensive staining, 1+ - weak staining, 0 - no staining. Scale bar, 50 µm.
Figure 3Left panel, growth of HCT116 VPS4B-/- shVPS4A cells as xenografts in mice in the presence or absence of doxycycline. Day 1 indicates the first day of doxycycline administration. n=9 for each group, each mouse bearing one tumor, ±SEM. Two-tailed unpaired t-test; ns - non-significant (p≥0.05), **p<0.01. Right panel, scatter plot representing end-point volumes of single xenografts. Bars represent means ±SEM.Immunoblotting analysis of VPS4A abundance in xenograft samples from untreated and doxycycline-treated mice (6 separate xenograft samples for each group). Lysates of HCT116 VPS4B-/- cells transfected with control or VPS4A-targeting siRNA marked VPS4A protein detection. GAPDH served as a loading control.Growth of HCT116 VPS4B-/- shCTRL#1 cells as xenografts in mice in the presence or absence of doxycycline. The arrow indicates the first day of doxycycline (Dox) administration. n=2 mice for each group, each mouse bearing two tumors, ±SEM.
Figure 4Heatmap visualizing expression of genes related to inflammatory response (left panel) and positive regulation of cell death (right panel) generated from the GO analysis of biological processes across different transfection conditions with at least 3 biological replicates.
Figure 5Immunoblotting analysis of the canonical and noncanonical branches of the NF-κB pathway and mediators of caspase-dependent cell death. Lysates of HCT116 VPS4B-/- cells were collected 66 h after transfection with siRNA (siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Lysates of HCT116 VPS4B+/+ and non-transfected HCT116 VPS4B-/- cells were used to monitor the basal pathway activity. Representative blot from 10 experiments is shown. NT- non-transfected; p-RelA - phospho-RelA; p-IκBα - phospho-IκBα; cl - cleaved caspases or PARP-1. GAPDH or vinculin served as loading controls.Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in (A). Data are means of 10 (phospho-Rel and cleaved caspase 7), 9 (p52, caspase 8 and cleaved caspase 9), 7 (phosphorylated and total IκB) or 5 (cleaved caspase 3) independent experiments. Error bars are SEM. Statistical significance was assessed using the following tests: one-sample t-test (caspase 8, cleaved caspases 9, 7 and 3, total and phospho-IκBα) or Wilcoxon signed rank test (phospho-RelA and p52); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Immunoblot showing induction of the NF-κB pathway (p52) and apoptosis (cleaved caspase 7) in xenograft samples described in Fig 3C. cl - cleaved caspase. Vinculin was used as a loading control.Analysis of the impact of RIPK1 inhibitor (necrostatin-1) or pan-caspase inhibitor (Q-VD-Oph) on cell viability of HCT116 VPS4B-/- cells transfected with siRNA (non-targeting siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Cell viability was assessed 72 h after siRNA transfection. Necrostatin-1 (50 µM), Q-VP-Oph (20 µM) or vehicle were added to the medium 48 h before viability assessment. Data are means of 5 independent experiments ±SEM. All values were normalized, viability of siCTRL-transfected and vehicle-treated cells was set as 100%. Two-tailed unpaired t-test; **p<0.01.
Figure 6Measurement of ATP released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.Measurement of HMGB1 released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.Microscopy images presenting cell surface calreticulin (green) in HCT116 VPS4B-/- cells 48 h after transfection with siRNA (non-targeting siCTRL#1 or targeting siVPS4A#2). As a positive control for detection of cell surface calreticulin, non-transfected cells (NT) were treated with 2 µM mitoxantrone for 24 h. In blue, DAPI staining. Scale bar, 15 µm.Flow cytometric analysis of calreticulin exposed on the cell surface of VPS4A-depleted HCT116 VPS4B-/- cells 66 h after siRNA transfection (siVPS4A duplexes: #2, #4 or #5 were used). Non-transfected (NT) or siCTRL#1-transfected cells served as negative controls. Left panel, percentage of cells positive for calreticulin in the population of live (DAPI-negative) cells, data are means of 4 independent experiments ±SEM. The Mann-Whitney U test; **p<0.01. Right panel, representative dot plot diagrams of flow cytometric analysis of cell surface-exposed calreticulin. Primary rabbit anti-calreticulin and control isotype IgG antibodies were used for staining, followed by secondary AlexaFluor 647-conjugated antibody.qPCR analysis of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage polarization markers in mouse bone marrow-derived macrophages (BMDMs) incubated for 24 h in conditioned media (CM) collected from control (siCTRL#1), Vps4a- and/or Vps4b-depleted CT-26 cells. For double Vps4a+b depletion various combinations of siVps4a#2 or #5 and siVps4B#3 or #4 duplexes were used. Data were normalized and are presented as the fold change of expression of a given M1 or M2 marker in BMDMs treated with CM from Vps4-depleted cells compared to its expression in BMDMs treated with CM from siCTRL#1-transfected cells (set as 1). Data are means of 4 independent experiments ±SEM. One-sample t-test; ns - non-significant (p≥0.05), *p<0.05, **p<0.01.
Figure 1(A) Stable RNAi screening of cancer-related genes was conducted to identify gene knockdowns that enhance reprogramming of MEFs into iPSCs. The graph shows the 10 shRNAs that were most strongly, positively selected in iPSCs compared to non-reprogrammed cells on day 14 of reprogramming (n=3 biological replicates).(B-E) Mouse embryonic fibroblasts (MEFs) were co-infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors. The co-transduced cells were sorted and were reprogrammed for 14 days: (B) Representative images and (C) quantification of alkaline-phosphatase-positive (AP+) iPSC colonies on day 14 of reprogramming by transfection of 4 reprogramming factors (=4F: Oct4, Sox2, Klf4, c-Myc = OSKM) or 3 factors (=3F: OSK). [n=3 biological replicates per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test analyzed by one-sided t-test with Holm-Sidak correction for multiple testing].Mouse embryonic fibroblasts (MEFs) were co-infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors. The co-transduced cells were sorted and were reprogrammed for 14 days: (D) Representative FACS profiles(E) percentage of Oct4-GFP+ iPSCs obtained after 14 days of reprogramming of MEFs from Oct4-eGFP mice using 4F-transduction on day zero that were infected with shRNAs targeting Tnfaip2 or a scrambled shRNA control [n=5 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test with Holm-Sidak correction for multiple testing].]. (F) mRNA of Tnfaip2 was measured by RT-qPCR at the indicated days of reprogramming by 4F-transduction on day zero [n=3 biological replicates; log-transformed data were not normally distributed (P>0.05 as per Shapiro-Wilk test), P value for the upregulation of Tnfaip2 on consecutive test days-9,-12 and -14 was calculated starting with the probability of maximum rank-based difference, i.e. having two groups of three data points perfectly separating, p1,b = 0.1 (bidirectional) and p1,u = 0.05 (unidirectional); the probability of finding a triple series of max. difference starting at some timepoint is Ptriple,tp = p1,b · p1,u · p1,u, and finding such a triple somewhere across the 5-step time series is Ptriple = Ptriple,tp + 2 · (1- p1,u) · Ptriple,tp = 0.0007.
Figure 2(C) RT-qPCR analysis of Smed-exoc3 mRNA expression of total RNA (whole body-derived) from exoc3(RNAi)-treated planarians compared to control-injected planarians on day-38 of the injection scheme [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test); data were analyzed by one-sided t-test].(D) Representative photographs of exoc3(RNAi)-treated planarians vs. control planarians at day-38 of the injection protocol as shown in panel B (n=4 experimental replicates with 6-10 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs depict the total number of animals showing the indicated phenotype.
Figure 3Planarians were injected with Smed-exoc3 dsRNA = exoc3(RNAi) or with control gfp(RNAi) injections. (A) The representative photographs show staining for phosphorylation of 10th serine residue of Histone 3 (H3S10p, a stem cell marker in planarians) at days-38 of the injection protocol (n=3 experimental replicates with 8-10 biological replicates per experiment, scale bar: 0.5 mm).exoc3(RNAi)-treated planarians versus gfp(RNAi)-treated controls were analyzed at the indicated time-points of the dsRNA injection protocol: (B) Quantification of H3S10p+ cells per mm2 in planarians at day-30, -34 and -38 of the injection protocol [n=3 experimental replicates with 5-7 biological replicates per experiment; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].FACS-based time-course analysis on changes in the fraction of somatic stem cells (X1-population) at indicated time points. Cells were obtained from whole body-trypsinized planarians of gfp(RNAi)-injected controls and exoc3(RNAi)-treated planarians and stained with the cytoplasmic dye Calcein-AM and the nuclear (DNA) dye Hoechst 33342: (C) Representative FACS profiles(D) quantification of the relative number of X1 cells [n=3 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].RT-qPCR analysis for makers of differentiating progenitor cells from the epidermal lineage on total (whole body derived) RNA from exoc3(RNAi)-treated planarians (E) Expression analysis of differentiation marker prog-2 on day-38 of the injection scheme [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test and analyzed by one-sided t-test],(F) Expression analysis of marker genes of differentiated cells (prog-1 and Smed-odc-1; on day-30, -34 and -38 of the injection scheme [n= 3 biological replicates per group and time point except for prog-1 expression at day 38, which includes 6 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons]. For Smed-odc-1 log-transformed mean centered group data were normally distributed and and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].
Figure 4Planarians were injected with exoc3(RNAi) or with control gfp(RNAi) injections. Organ phenotypes were analyzed on day-38 of the injection protocol. Organ homeostasis was analyzed by staining against specific markers: (A) The structure of the brain/central nervous system (CNS) was analyzed by staining against synapsin (3C11). The cartoon represents the phenotypic change (upper panel). Representative photographs are shown in the lower panel (3 experimental replicates with 2-3 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype. There was a significant increase in the occurrence of atrophic cephalic ganglia and sensory neuron loss in exoc3(RNAi)-treated planarians vs. controls (cg: cephalic ganglia; sn: sensory neurons).(B) Planarian visual system was analyzed by staining against VC1. Schematic cartoon (upper panel) and representative images (lower panel) are shown (3 experimental replicates with 3 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype. Arrows point to photosensitive cells (ps); pigmentary cells (pc) and to the optic chiasm (oc).
Figure 5RNA-seq was conducted on freshly isolated X1 cells from exoc3(RNAi)-treated compared to X1 cells from gfp(RNAi)-treated planarians on day-38 of the injection protocol. The histograms depict the overlap (blue) of DEGs in X1 cells of Smed-exoc3-depleted planarians versus controls (n=3 biological replicates; adjusted P< 0.05) with genes expressed in the indicated sample sets of (A) neoblast stem cells(B-E) The heat maps depict the intersection of DEGs with strong expression signals (log2 fold change > 0.5 or < -0.5) discovered in X1 cells of exoc3-depleted planarians versus controls (n= 3 biological replicates) with the indicated, previously published data sets on gene expression profiles in (B-D) neoblast cells or (E) differentiating progenitor cells of epidermal lineage. The colour scale represents the gene-wise z-score calculated from normalized gene expression levels while purple and green indicate upregulated and downregulated genes in X1 cells of exoc3-depleted planarians versus controls, respectively.RNA-seq was conducted on freshly isolated X1 cells from exoc3(RNAi)-treated compared to X1 cells from gfp(RNAi)-treated planarians on day-38 of the injection protocol. The histograms depict the overlap (blue) of DEGs in X1 cells of Smed-exoc3-depleted planarians versus controls (n=3 biological replicates; adjusted P< 0.05) with genes expressed in the indicated sample sets of (F) differentiating progenitor cells of epidermal lineage from the indicated publications while the sum of overlapping (blue) and set-exclusive (grey) genes represents the total number of genes of a cluster that were analyzed in this study.
Figure 6Planarians (S. mediterranea) were injected with exoc3(RNAi) or with control gfp(RNAi). (B) Representative photographs of morphology of exoc3 knockdown planarian vs. control planarian on days-29, -31 and -37 of the protocol Number ratios in the photographs indicate the photographically represented phenotypes.Organ regeneration was analyzed by immunostaining of different organ compartments in regenerating exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated control on day 37 of the protocol (C) Eyes were analyzed by staining against VC1. Representative images are shown (3 experimental replicates with 4-5 biological replicates per experiment). Arrows point to photosensitive cells (ps); pigmentary cells (pc) and to the optic chiasm (oc). Number ratios in the photographs refer to planarians with regenerative defects for the indicated phenotype (scale bar: 0.5 mm).Organ regeneration was analyzed by immunostaining of different organ compartments in regenerating exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated control on day 37 of the protocol (D) Representative photographs of staining with planarian pan-neural marker 3C11 (alias Synapsin) marking cephalic ganglia and sensory neurons Number ratios in the photographs refers to planarians with regenerative exhibiting the photographically represented phenotype, characterized by underdeveloped bilobed cephalic ganglia and atrophy of sensory neurons in exoc3(RNAi)-treated planarians versus controls (scale bar: 0.5 mm, cg: cephalic ganglia; sn: sensory neurons).