EMBO/SourceData huggingface dataset:
Figure 2A Flow cytometry analysis of neutrophils in hearts and representative immunostainings for neutrophils in the infarct area, identified as Ly6G-positive (5x and 20x magnifications). Two-way ANOVA followed by Bonferroni post-hoc test; n=5 mice for no MI, n = 5 for 12h post MI, n = 3 for 24h post MI and n = 3 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0161 (12h MI), *P = 0.003 (24h MI).B Flow cytometry analysis of neutrophils in bone marrow. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice for no MI, n = 5 for 12h post MI, n = 4 for 24h post MI and n = 4 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0471 (12h MI), *P = 0.0035 (24h MI).C Blood counts of granulocytes. Two-way ANOVA followed by Bonferroni post-hoc test; n= 9 mice for no MI, n = 10 for 12h post MI, n = 13 for 24h post MI and n = 10 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0364 (24h MI).D Flow cytometry analysis of neutrophils in hearts and bone marrow under inverted light cycle conditions. Two-way ANOVA followed by Bonferroni post-hoc test; n = 3 mice for no MI in both ZT groups, n = 5 for ZT5 and n = 6 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0147 (24h MI).E Representative gating strategy for GMPs in the bone marrow, identified as identified as lineage negative (CD11b-, Gr1-, B220-, CD3- and Ter119-) and Sca-1-, c-kit+, CD16/32+, CD34+. Flow cytometry quantification of GMP in the bone marrow. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for no MI and 24h post MI in both groups; ZT5 vs. ZT13: *P = 0.0077 (no MI), *P = 0.0013 (24h MI).F CXCL12 levels in bone marrow lavage. Two-way ANOVA followed by Bonferroni post-hoc test; n = 7 mice for no MI and n = 6 for 24h post MI in both groups; ZT5 vs. ZT13: *P = 0.0077 (no MI), *P = 0.0013 (24h MI).G Plasma levels of pro-inflammatory cytokines and chemokines. Two-way ANOVA followed by Bonferroni post-hoc test; n = 11 mice for no MI in both ZT groups, n = 7 for ZT5 and n = 8 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0271 (CXCL12, no MI), *P = 0.0108 (TNFα, 24h MI), *P = 0.001 (G-CSF, 24h MI), *P = 0.005 (CXCL1, 24h MI), *P = 0.0005 (CXCL2, 24h MI), *P = 0.0016 (CCL3 24h MI), *P = 0.0144 (CCL5, 24h MI).
Figure 3A Permanent LAD occlusion was perfomed at ZT5 or ZT13. TTC staining (white, infarct; red, vital myocardium) and quantification of infarct size normalized to the left ventricle (LV). Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0041.B Plasma troponin I levels 24 hours after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0007.C Flow cytometry analysis of dead cardiomyocytes (CD45-, zombie+) 24 hours after MI. Student`s t-test; n = 3 mice in both groups; *P = 0.0072.D Survival rates after MI and cause of death. Log-rank test; n = 87 mice in both groups; *P = 0.0006.E Myofibroblasts within infarcts were quantified by alpha smooth muscle actin (αSMA) staining as ratio between stained and total area of random fields. Student`s t-test; n = 4 mice in both groups; *P = 0.0134.F Masson´s trichome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total LV (*P = 0.0095) as well as LV anterior wall thickness (*P = 0.0068) 7 days after MI. Student`s t-test; n = 6 mice for ZT5 and n = 7 for ZT13 MI.G Analysis of relative collagen type I content identified by Sirius Redstaining 7 days after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0175.H Echocardiography assessment of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV). Two-way ANOVA; n = 6 mice for no MI for both groups, n = 6 for ZT5 and n = 9 for ZT13 at 72h post MI, n = 8 for both groups at 7d post MI and n = 7 for ZT5 and n = 5 for ZT13 14d post MI; ZT5 vs. ZT13: *P = 0.0001 (EF, 3d), *P = 0.0042 (EF, 7d), *P = 0.0121 (EF, 14d); *P = 0.0253 (ESV, 3d), *P = 0.0421 (ESV, 7d), *P = 0.0005 (ESV, 14d); *P = 0.0053 (EDV, 14d).
Figure 4B Flow cytometry analysis of cardiacneutrophils 24 hours after ZT13 MI. The dotted line indicates cardiacneutrophil counts 24h after ZT5 MI, shown in Fig. 2A. Student`s t-test; n = 3 mice for isotype and n = 5 for Ly6G injected mice; *P = 0.0004.C Plasma TNFα levels 24 hours after ZT13 MI. Student`s t-test. n=8 independent samples for isotype and n=6 for Ly6G injected mice; *P = 0.0019.D Infarct size relative to left ventricular area (LV) and plasmatroponin I levels 24 hours after ZT5 or ZT13 MI. Student`s t-test; For infarct size n = 3 mice in both groups for ZT5 and n = 4 mice for isotype and n = 3 mice for Ly6G at ZT13; Isotype vs. Ly6G: *P = 0.0158 (ZT13). For troponin levels n = 8 mice for isotype and n = 6 mice for Ly6G with ZT13 MI; *P = 0.0012.E Masson's-trichrome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total area of the LV (*P = 0.0084); morphometric quantification of the LV anterior wall thickness (*P = 0.0027) 7 days after ZT13 MI. Student`s t-test; n = 5 mice for isotype and n = 4 mice for Ly6G.F Analysis of collagen type I fibers within infarcts identified by Sirius Red staining 7 days after ZT13 MI. Student`s t-test; n = 5 mice for isotype and n = 4 mice for Ly6G; *P = 0.0375.G Echocardiography measurement of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV) before and after ZT13 MI. Two-way ANOVA; n = 6 mice for no MI in both groups, n = 9 for isotype and n = 5 for Ly6G at 72h post MI, n = 8 for isotype and n = 4 for Ly6G at 7d post MI. Isotype vs. Ly6G: *P = 0.0001 (EF, 3d), *P = 0.0227 (EF, 7d); MI *P = 0.0133 (ESV, 3d), *P = 0.0004 (ESV, 7d); *P = 0.0017 (EDV, 7d).
Figure 5A Representative flow cytometry analysis and quantification of CXCR2highneutrophils in the blood after ZT5 and ZT13 MI. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for all time points in both groups; ZT5 vs. ZT13: *P = 0.0001 (4h), *P = 0.0001 (8h), *P = 0.0447 (12h).B Percentage and mean fluorescence intensity (MFI) of CXCX2 expression by cardiacneutrophils 12h post-MI after ZT5 and ZT13 MI. Student`s t-test; n = 4 mice in both groups; *P = 0.0001.C Percentage of CXCR2highneutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0005 (ZT13).D Flow cytometry quantification of neutrophils in hearts 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0079 (ZT13).E Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0002 (ZT13).F Mean fluorescence intensity (MFI) of CXCX2 expression at ZT5 in the blood 24h after MI in wildtype (WT) and CXCR2 KO mice. Student`s t-test. N = 6 mice in both groups; *P = 0.0001.G Percentage of CXCR2hi bloodneutrophils 24h after ZT5 or ZT13 MI in WT and CXCR2 KO mice. Two-way ANOVA followed by Bonferroni post-hoc test; for ZT5 n = 6 mice in both groups, for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice; WT vs. CXCR2 KO: *P = 0.0001 (ZT13).H Flow cytometry quantification of neutrophils in hearts 24h after MI in ZT5 and ZT13 operated WT and CXCR2 KO mice. Two-way ANOVA followed by Bonferroni post-hoc test; for ZT5 n= n = 6 mice in both groups, for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice; WT vs. CXCR2 KO: *P = 0.0461 (ZT13), ns = not significant.
Figure 6B Percentage of CXCR2hineutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0.0046 (ZT13); ZT5 vs. ZT13: *P = 0.0153 (DMSO).C Flow cytometry quantification of cardiacneutrophils 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0.0006 (ZT13); ZT5 vs. ZT13: *P = 0.0001 (DMSO).D Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0,0486 (ZT13).
Figure 1GPS reporter cells expressing peptide libraries ending with a random (X) or Gly (G) residue were treated with dominant-negative (DN) cullins and analyzed. Data are presented as mean ± standard deviation from nine replicates. GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC-box proteins and analyzed. VHL and FEM1C serve as unrelated BC-box protein controls. Data are presented as mean ± standard deviation from nine replicates.
Figure 2Comparisons of protein stability among wild-type protein and the mutants indicated on the right.
Figure 3(A) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations. The number on each graph denotes the left-tailed p-value of the -1 residue. CT-inside and CT-outside represent membrane proteins with their C-termini facing the cytosol or extracellular space, respectively.(G) Confocal images of U2OS cells expressing MTSGLUL-GFP proteins with or without a C-terminal diGly degron. MitoTracker staining shows mitochondria. Scale bar = 20 µm. (H) Confocal images of U2OS cells expressing MTSGLUL-GFP-diGly degron fusion proteins co-stained with MitoTracker, and with or without DNCul2 treatment. Scale bar = 20 µm.
Figure 4Stability analysis of indicated proteins in cells treated with shRNAs against various BC-box proteins. Since GFP was tagged at the protein N-terminus and signal peptide and MTSs need to be exposed to be functional, all tested proteins were mislocalized. Protein stability analyses of wild-type (WT) and mutant proteins lacking their extreme terminal Gly (∆G) or having it masked by adding Leu (+L) in cells with or without BC-box protein knockdown (C) or overexpression (D).Fractionation and Western blot analysis of wild-type and Flag-tagged PDP2 (top), and wild-type and MTS-deleted (∆MTS) MRPL28 (bottom). Flag-PDP2 and ∆MTS MRPL28 were mislocalized. W, C, and M denote whole cell lysates, cytosol fraction and mitochondrial fraction, respectively. Tubulin and VDAC serve as fractionation quality controls.Protein abundance analysis of HA-tagged wild-type or mutant secretory proteins with their signal peptide deleted (∆SP) in cells with or without KLHDC3 or APPBP2 expression. The HA epitope was inserted immediately after the signal peptide (SP), which had no impact on protein secretion.
Figure 5Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2, DNCul2 or IMP-1088 treatments. IMP-1088 inhibits NMTs. C and M denote cytosol and mitochondrial fractions, respectively. Tubulin and VDAC serve as fractionation quality controls. The relative abundance of cytosolic MIC19/tubulin was normalized to that of lane 1 and is indicated under each lane in red. Another replicate of this experiment is shown in Fig. EV3B.Abundance comparison of GFP N- or C-terminal tagged with the wild-type or mutant N-terminal (aa. 1-24) or C-terminal (a.a. 218-227) motif of MIC19 in cells with indicated treatments. Data from sh#1 is shown here.
Figure 6Fractionation analysis of wild-type HA-MIC19 in U2OS cells treated with IMP-1088 and with or without inhibition of ZYG11B or KLHDC2. The normalized relative abundance of cytosolic HA-MIC19/tubulin is indicated under each lane in red.
Figure 7GST pull-down assay using cells expressing GST or GST-tagged wild-type or mutant KLHDC2 and HA-tagged SDE2, FAU or TUG.
Figure 8Relative stability analysis of GAPDH C-terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays. The -3, -4 or -5 residue of SARS-CoV NSP proteins was mutated to Ala (A) as indicated. The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2. Data are presented as the mean value of GFP/RFP +/- standard deviation from triplicate experiments.
f1a, Wild-type (WT) and Park2-/- BMDMs were infected with mCherry-expressing M. tuberculosis (Mtb) for 4 h and immunostained using anti-parkin antibodies.b, Wild-type BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for polyubiquitin.b, Wild-type BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for polyubiquitin. c, Quantification of ubiquitin-positive M. tuberculosis from b. Results are means ± s.e.m. of three independent experiments (**P < 0.001, paired Student's t-test).d, U937 human macrophages expressing a scrambled shRNA (Control) or one of two different shRNAs targeting PARK2 (shRNA no. 1, shRNA no. 2) were infected with mCherry-expressing M. tuberculosis for 12 h and immunostained for polyubiquitin.d, U937 human macrophages expressing a scrambled shRNA (Control) or one of two different shRNAs targeting PARK2 (shRNA no. 1, shRNA no. 2) were infected with mCherry-expressing M. tuberculosis for 12 h and immunostained for polyubiquitin. e, Quantification of ubiquitin positive M. tuberculosis from d, results are means ± s.e.m. of three independent experiments (**P  0.005, Student's t-test). f, The parkin and actin expression in cells from e was determined by western blotting.g, Park2−/− BMDMs were transduced with lentivirus expressing BFP (−), wild-type parkin, or two separate mutant parkin alleles (T240R, P437L). Cells were infected with M. tuberculosis for 4 h and ubiquitin and M. tuberculosis co-localization was quantified and expressed relative to control BMDMs. Results are means ± s.e.m. of three independent experiments (**P  0.005, paired Student's t-test). h, The parkin and actin expression in cells from g was determined by western blotting.
f2a, BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained using linkage-specific ubiquitin antibodies. b, Quantification of K63 and K48 ubiquitin-positive M. tuberculosis from a.c, BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for NDP52, p62, phospho-TBK1 or NBR1. d, Quantification of co-localization from c (n = 3 per group, **P  0.001). All errors, s.e.m.
f3a, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 4 h and immunostained for LC3 (LC3B antibody) or ATG12. b, Quantification of results from a (n = 3 per group, **P  0.001). c, Western blot analysis of LC3 (LC3B antibody) from cell lysates from a.d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1.d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1. e, Quantification of results from d expressed relative to control BMDMs (n = 3 per group, **P  0.001).f, BMDMs were infected with M. tuberculosis and colony-forming units (c.f.u.) at t = 0 and t = 16 h were determined by plating. Results were normalized to t = 0 (n = 3 per group, *P < 0.02).g, RAW264.7 macrophages transduced with lentivirus expressing green fluorescent protein (GFP) or parkin were infected with M. tuberculosis and c.f.u. were determined after 16 h. Results were normalized to GFP-expressing cells (n = 3 per group, **P < 0.0076).h, U937 human macrophages expressing either scrambled or PARK2 shRNAs were infected with M. tuberculosis for 36 h and c.f.u were determined. Results were normalized to t = 0 (N = 3 per group, *P< 0.02). All errors, s.e.m.
f4a, WT and Park2-/- mice were infected with wild-type M. tuberculosis by aerosol and lung bacterial burdens were determined by plating (means ± s.d., n = 5 per group, *P < 0.02 by Student's t-test).b, Enumeration of liver and spleenc.f.u. from mice infected in a, 21 days post-infection (means ± s.d, n = 5 per group, *P < 0.03 by Student's t-test).c, Survival of M. tuberculosis-infected wild-type and Park2-/- mice (n = 10, **P < 0.001 by log-rank test).d, Immunohistochemistry staining of lung sections from infected mice21 days post-infection, scale bar, 50 µm.e, Immunohistochemistry staining of lung sections from a human patient with active tuberculosis. Scale bars, 500 µm (×4), 50 µm (×40).f, Confocal microscopy images of humanlung sections from e immunostained for M. tuberculosis and parkin; DNA was visualized using DAPI (4′,6-diamidino-2-phenylindole). Scale bars, 25 µm.g, Mice were infected with wild-type L. monocytogenes by i.p. injection and bacterial burdens in livers and spleens were determined by plating (means ± s.d., n = 7 per group, *P < 0.04 by Student's t-test).
f5a, Wild-type and two independent parkin-deficient D. melanogaster lines (parkc00062, parkf01950) were infected with L. monocytogenes by anterior abdomen injection. Atg8 processing was monitored in whole-fly protein lysates by western blotting.b, L. monocytogenes c.f.u. from a were determined by plating (means ± s.d., n = 3-5, **P < 0.001 by Student's t-test).c, Survival of D. melanogaster was determined (n = 33, **P < 0.001 by log-rank test).d, e, Flies were infected with S. enterica serovar Typhimurium and bacterial c.f.u. (d) (means ± s.d., n = 3-5 per group, **P  0.009 by Student's t-test) and fly survival (e) were determined (n = 59, **P  0.001 by log-rank test).f, Survival of flies infected with M. marinum (n = 37, **P  0.0045 by log-rank test).
Figure 1Immunofluorescence images of U2OS cells expressing FLAG-HA-FAM134 after 24hrs doxycycline induction stained for HA (green) and endogenous calnexin (CANX; red). Scale bar: 20μm.
Figure 2Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) purified GST, wild type GST-LC3s, GST-GABARAPs, GST-Ubiquitin (Ub), GST-Tetra-Ubiquitin, A representative Ponceau staining is shown.Lysates of HEK293T cells transiently transfected with HA-tagged wild-type and wild-type or ∆LIR mutant FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) A representative Ponceau staining is shown.Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) using GST-LC3B and GST- LC3B F52A-V53A as baits. A representative Ponceau staining is shown.Immunofluorescence images of U2OS expressing wild-type or ∆LIR mutant FLAG-HA-FAM134 proteins after 24hrs doxycycline induction under different conditions (basal=DMSO, Baf.A1=2h 200nM Bafilomycin A1, EBSS=2h starvation in EBSS) Scale bar: 10μm; staining against FAM134 (HA; green) and endogenous LC3B (red).
Figure 3Small sections of sample pictures of U2OS cells stably expressing the ER-phagy reporter ssRFP-GFP-KDEL. Pictures were obtained by the IncuCyte® S3 (10x) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time. Scale bar 25µm. Time lapse of representative total image views can be found in Movie EV7 and Movie EV8.Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction. Autophagy flux was induced and blocked by treatment with Torin1 (8hrs) and Bafilomycin A1 (Baf.A1, 8hrs), respectively. n=3
Figure 4Representative Western blot for Fam134 proteins in single knockout mouse embryonic fibroblasts (MEFs).Western blot for Fam134 proteins in indicated MEFs, expressing the ER-phagy reporter ssRFP-GFP-KDEL and being reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein.
Figure 5Profile plots showing the Log2 LFQ intensity (G) and representative immunofluorescence (H) of Climp63 (green) in wild-type and Fam134 knockout MEFs.Profile plots showing the Log2 LFQ intensity (I) and representative immunofluorescence (J) for collagen I (red) expression in wild-type and Fam134 knockout MEFs. Scale bar: 10μm. Inset scale bar: 5μm. Nuclei were stained with Hoechst 33342.
Figure 6Electron microscopy of wild-type and Fam134 single knockout MEFs (ER=endoplasmic reticulum). Scale bar: 500nm.Immunofluorescence images stained for endogenous Calnexin (Canx) or Climp63 in wild-type (WT), Fam134 knockout MEFs, and Fam134 knockout MEFs reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein. Scale bar: 10μm. Inset scale bar: 5μm.Representative Western blots of total cell lysate derived from MEFs used in (B) and detected for endogenous Fam134 and Actin, respectively.
Figure 7Representative Western Blot of Collagen I in wild-type MEFs and Fam134 knockout MEFs reconstituted for the respective wild-type (WT) or ∆LIR mutant Fam134 protein. Vinculin has been used as reference for ratio calculation. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. of n=3 biological experiments and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001.Representative Western Blot (D) and the relative bar plot (E) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆LIR Fam134a protein.Representative Western Blot (F) and the relative bar plot (G) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a and Fam134c. Representative Western Blot (H) and the relative bar plot (I) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a and Fam134b.Representative Western Blot (J) and the relative bar plot (K) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b. Representative Western Blot (L) and the relative bar plot (M) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c.
Figure 1C. Confocal images of MDA-MB-231 cells transfected with ZsGreen or ZsRASSF1C grown in 3D-collagen matrix. Cells were imaged and stained with Phalloidin-568 (red), pMLCII/Alexa‑633 (false‑coloured green) or imaged for ZsGreen (false-coloured white). Scale 20 μm.D. Single cell morphology assay of MDA-MB-231 cells cultured in 3D-collagen and analysed for morphology 24 hours after transient knock-down using either a control sequence (NT, non-targeting) or a sequence targeting the RASSF1 locus. RASSF1 knock-down marks a reduction in rounded cells. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Figure 2C. Western blot analysis of proteins in MDA-MB-231 cells transiently transfected with siRNA targeting a control sequence (NT) or targeting RASSF1. GAPDH was used as a loading control.D. Immunoprecipitation from MDA-MB-231 transiently transfected with control (pcDNA3) or HA‑RASSF1C plasmid. Pull‑down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies.H. Quantification of rounded versus elongated cells in single cell morphology assay in 3D‑collagen indicating the degree of mesenchymal-amoeboid transition of MDA-MB-231 cells when RASSF1C or the described mutants are expressed. I. Representative confocal images show elongated or rounded cells per field of view of MDA‑MB-231 cells transfected with DsRed, DsRASSF1C, DsRASSF1C-R197W or DsRASSF1C-R199F, grown in 3D‑collagen and stained with Phalloidin-568 (red) for F‑actin and pMLCII/Alexa 633. Scale bars represent 10 μm.