Calcium condensation of Tat/pDNA complexes into unhazardous supply nanoparticles
To cut back the toxicity and at similar time to enhance the gene supply effectivity of Tat/pDNA complicated, calcium chloride was used as a compact agent to condense the particles of Tat/pGL3 complicated. Gel retardation assay proven that Ca2+ addition (last conc. 113 mM) effectively condensed Tat/pGL3 or Tat/pGL3-DiYO-1 complexes into nanoparticles even when the N/P ratio reached to 1 (Fig. 1A, B). This can be a vital enchancment from our earlier statement that Tat-facilitated pDNA compact formation solely occurred when N/P ratios greater than 5 . Condensation capability of Ca2+ addition was constant when the N/P ratio of Tat/pDNA complexes elevated from 1 to twenty. Condensing stability of Tat/pDNA-Ca2+ nanoparticles was decided by DNase I safety assay. Whole resistance to DNase I motion was noticed for Tat/pGL3-Ca2+ nanoparticles induced at N/P ratios not lower than 1 (Fig. 1C). DLS (dynamic gentle scattering) measurements confirmed that formulated Tat/pGL3-Ca2+ nanoparticles offered weak optimistic ζ potential (common 3.8 mV), and possessed small particle measurement (252–512 nm) at N/P ratio of 10 (Fig. 1D. Further file 1: Desk S1. Further file 2: Fig. S1). These outcomes are just like earlier findings [11, 30], highlighting the impact of Ca2+ addition to Tat/pDNA complexes induced substantial lower within the particle measurement and competitively inhibit the amine/phosphate interplay. This synergic impact could be attributed to the “gentle” condensation capability of Ca2+ to Tat/pDNA complexes, as a result of outlined focus vary of calcium interactions with each amines (polycations) and phosphates (DNA) can management particle measurement [11, 31, 32]. The uneven size-distribution of Tat/pGL3-Ca2+ nanoparticles is suitable with the outcomes from AFM and TEM checking pictures, and irregular granules with uneven morphology have been continuously noticed (Fig. 1E–H). Excessive optimistic ζ potential and small diameter of Tat/pGL3-Ca2+ nanoparticles have been reached with enhanced N/P ratios (Further file 1: Desk S1). With out Ca2+ addition, we solely noticed massive, free complexes between Tat peptide and pDNA , whereas small, compact Tat/pDNA nanoparticles have been fashioned after Ca2+ addition (Fig. 1E–H. Further file 1: Desk S1).
In comparison with earlier formulation [6, 7], including Ca2+ on this work led to high-efficient supply of Tat/pDNA complexes with out obvious toxicity (Figs. 2, 7E, Further file 2: Fig. S2, S13). Much less apoptosis-induced in OC cells and negligible physique weight or visceral modifications in OC-bearing mice have been noticed after therapy with Tat/pDNA-Ca2+ nanoparticles. Noticeably, improved cell survival was noticed after including Ca2+ into Tat/pDNA complicated (Further file 2: Fig. S2C-D), and solely high-concentration (20ug/mL, NP = 10), long-term (14 h) publicity of Tat/pGL3-Ca2+ nanoparticles to cells can set off necrotic apoptosis (Further file 2: Fig. S2A-B). It was reported that Ca2+ addition (> 30 mM) can scale back aggregation and yield of extra monodisperse lipoplexes . We noticed agglomeration of Tat/pDNA complexes with out CaCl2, whereas Tat/pDNA-Ca2+ nanoparticles with optimum CaCl2 focus (113 mM) have been comparatively steady in mouse serum and serum-contained tradition media (Further file 2: Fig. S3). This stability was additionally demonstrated by the heparin displacement assay, as a result of exposing the Tat/(SYBR Inexperienced)-pDNA-Ca2+ nanoparticles to the extremely negatively charged heparin yielded a rise within the fluorescence sign with enhance in heparin focus (Further file 2: Fig. S4). Secure physique weight of mice and no visceral toxicity in OC-bearing mice (Further file 2: Fig. S13, Fig. 7E), point out that the formulated Tat/pDNA-Ca2+ nanoparticles are secure. These outcomes demonstrated that Tat/pDNA complexes have been tightly condensed into small, unhazardous supply nanoparticles by Ca2+ addition.
Cell-cycle-dependent macropinocytic uptake of Tat/pDNA-Ca2+ nanoparticles
Macropinocytosis was reported as a dominant uptake route for Tat-based gene supply vectors [6, 7, 16, 34,35,36]. Right here, we discovered that Tat/pGL3-Ca2+ nanoparticles primarily utilized macropinocytosis for uptake in OC cells. Utilizing numerous endocytic markers as indicated within the literatures, confocal imaging confirmed that Tat/pGL3-Ca2+ nanoparticles weren’t co-localized with the clathrin-mediated endocytosis (CME) marker Tfn-AF647 and lipid raft marker CTxB, however co-localized with the macropinocytosis marker Dextran (Fig. 2A, Further file 2: Fig. S5A, S6). SKOV3 cells pretreated with CME inhibitor CPZ, or caveolae-mediated endocytosis (CvME) inhibitor filipin, or cholesterol-depleting reagent MBCD didn’t considerably inhibit the uptake of Tat/pGL3-Ca2+ nanoparticles. In distinction, when cells have been pretreated both with the macropinocytosis inhibitor EIPA or with CyD, the uptake of nanoparticles was considerably inhibited (Further file 2: Fig. S5B). These outcomes have been just like our earlier observations [6, 7, 27]. The phenomenon that Tat/pDNA-Ca2+ nanoparticles most popular macropinocytic uptake in OC cells might be defined by: (1) since macropinocytic capability is decided by the properties of cells [14, 15], intensified macropinocytosis of Tat/pDNA complicated was induced in OC cells with sure mutations . (2) Constructive-charged, botryoid-shaped nanoparticles enter tumor cells by way of macropinocytosis extra effectively than negative-charged, shape-defined nanoparticles [17, 37]. Our Tat/pDNA-Ca2+ nanoparticles with irregular and uneven morphology (Fig. 1E, H) largely manipulate macropinocytosis to favor their intracellular supply; (3) Cationic CPPs binding with anionic glycosaminoglycans (GAGs) on cell floor all the time stimulate Rac activation, which induces actin polymerization, lamellipodia formation and subsequent macropinocytosis-initiation [20, 38, 39]. Extra Tat peptide inside Tat/pDNA-Ca2+ nanoparticle (NP > 10) are vulnerable to stimulate macropinocytosis boosted in OC cells (Fig. 2, 4); (4) Most cancers cells present process blebbishield emergency program exhibit sturdy macropinocytosis , the pro-apoptotic standing of OC cells stimulated by Tat/pDNA-Ca2+ nanoparticles (Further file 2: Fig. S2) might additional promote the macropinocytosis in these cells. As well as, we additionally noticed macropinocytic distinction between Skov3 and Cos7 cells (Fig. 2Ok, L, Fig. 4A–D), suggesting the cell-specific uptake of Tat/pDNA-Ca2+ nanoparticles [4, 32], which is of significance for design therapy of various cell varieties.
Totally different from the prevailing claims that pinocytosis is shut down throughout mitosis [41,42,43], we discovered right here that macropinocytosis was decreased however not utterly arrested in cell-cycle M-phase. Confocal microscopic inspecting the co-uptake of Tat/pGL3-Ca2+ nanoparticles with Dextran in unperturbed cells confirmed that they have been nonetheless internalized by way of macropinocytosis in several sub-phases of cell-cycle (Fig. 2A–H). Macropinocytic uptake of Tat/pGL3-Ca2+ nanoparticles appeared continuously shutdown in cells at I-phase (interphase) (Fig. 2B–D). To make clear these variations, we synchronized cells at distinct sub-phases of cell-cycle by chemotherapeutics pretreatments (Fig. 2E). Confocal microscopic inspecting the co-uptake of Tat/pGL3-Ca2+ nanoparticles with Dextran in arrested cell-cycle phases confirmed that their macropinocytosis have been markedly elevated in GCB(gemcitabine)-induced S-phase and endured in PTX(paclitaxel)-induced M-phase (Fig. 2F–H). Figuring out these variations by fluorescence quantification assay additionally confirmed the improved co-uptake of Tat/pGL3-Ca2+ nanoparticles with Dextran in cells at S- and M-phases (Fig. 2I, J). Addition of macropinocytosis inhibitor EIPA (5-(N-ethyl-N-isopropyi)-amiloride) completely reversed this enhancement (Further file 2: Fig. S6A, B). Additional checking the impression of Tat/pDNA-Ca2+ nanoparticles on cell-cycle distribution discovered that sub-phases of asynchronous or synchronized cells weren’t considerably altered after incubating with Tat/pGL3-Ca2+ nanoparticles (Further file 2: Fig. S7). Moreover, enhanced transgene expression of Tat/pGL3-Ca2+ nanoparticles in S- or M-phase cells and transduction-inhibition by EIPA co-incubation have been noticed (Fig. 2Ok, L, Further file 2: Fig. S6C). These outcomes counsel that macropinocytosis and expression of Tat/pDNA-Ca2+ nanoparticles have been endured in cell-cycle M-phase and markedly elevated in cell-cycle S-phase.
Earlier research confirmed that a number of mobile uptake pathways (caveolar and clathrin-mediated endocytosis) do happen throughout regular mitosis when checking asynchronous cells underneath superior microscopy (e.g. Electron microscopy) [44,45,46]. Contemplating solely 0.5–2% of mammalian cells present process mitosis and residual endocytosis are devoted to the turnover of plasma membrane and particular receptors throughout the successive course of (prophase, metaphase, anaphase, telophase and cytokinesis) [42, 47], re-activated macropinocytosis of Tat/pGL3-Ca2+ nanoparticles in partial cell inhabitants at M-phase are logical (Fig. 2B–H). Moreover, upregulated expression of macropinocytosis driver Arf6 at M-phase (Fig. 3B) additionally means that reactivated macropinocytosis is liable to assist plasma membrane transforming when cells present process division. Notably, the discovering right here is that macropinocytosis of Tat/pGL3-Ca2+ nanoparticles was extremely upregulated in GCB-induced S-phase cells (Fig. 2B–H). To our data, that is the primary statement that macropinocytosis peaked up at particular phases of cell-cycle. In vivo knowledge with enhanced supply and killing effectivity of Tat/TF-Ca2+ nanoparticles in GCB-administrated group additionally confirmed this discovering (Figs. 7C, D, 8A, B). Little is thought concerning the components accounting for this distinction phenomenon. As intact nuclear membrane and undersized nuclear pore complicated (10–26 nm) are not possible to help the nuclear uptake of Tat/pDNA-Ca2+ nanoparticles when transfection was carried out at this part, probably components could also be from sure levels of macropinocytosis. Excessive macropinocytosis of Tat/pDNA-Ca2+ nanoparticles however low Arf6 expression at S-phase seems paradoxical (Fig. 2F–H, Fig. 3B). Since Arf6 can regulate macropinocytosis and likewise act on the terminal stage of cytokinesis [48, 49], extremely expression and accumulation of Arf6 at M-phase is helpful for its localizing at cleavage furrow and midbody in order to complete the cytokinesis course of. Though activated Arf6 (GTP binding) is required for cell floor recycling of short-lived macropinosome, inactivation of Arf6 (GTP hydrolysis) is important to intracellular macropinosome trafficking [31, 50], due to this fact, low expression of Arf6 at S-phase contributes to energetic macropinocytosis is logical. As well as, Tat-based nanoparticle sure with cell floor GAGs is an important prerequisite for his or her uptake . As a result of the structural and quantitative variations of GAGs (particularly on the cell floor) throughout cell-cycle are nonetheless debated [33, 51], it’s laborious to narrate macropinocytosis kinetics to the expression profile of GAGs within the cells, but.
GRP75 pushed cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles
Seeing that cell-cycle and endocytosis-transition are synchronously regulated by moonlighting chaperones [24, 52, 53], we subsequent decided whether or not mitochondrial moonlighting chaperone GRP75 appearing on the cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles. Cyclebase-based predication confirmed that high-expression of GRP75 continuously appeared at S-phase and G2(hole part 2)/M boundary. Excessive-expression of GRP75-bound mitotic kinase MPS1 was primarily distributed from S- to M-phase. Nonetheless, high-expression of GRP75-bound, MPS1-recruited mitochondrial gatekeeper VDAC1 was primarily offered at S-phase. Remarkably, high-expression of macropinocytosis driver Arf6 was scarcely distributed at S-phase, G2/M boundary and early G1-phase (hole part 1) (Fig. 3A). Western blot evaluation of cell fractions confirmed that centrosome-associated GRP75 and MPS1 have been extremely enriched at S-, G2-, and M-phases than that at G1-phase. In distinction, centrosome-associated VDAC1 and Arf6 have been sharply diminished at these phases (Fig. 3B). Unexpectedly, complete lysate-derived and cytoplasmic VDAC1, Arf6 have been diminished at S-phases, and no vital expression-difference of GRP75 and MPS1 was detected. These outcomes counsel that GRP75 and MPS1 co-enriched at centrosome from cell-cycle S- to M-phase.
GRP75 was proven being related to duplicated centrosomes, phosphorylated by Mps1 on Thr62 and Ser65, and suggestions super-activated Mps1 in HeLa-1 and U2OS cells [54, 55]. To find out whether or not it may moonlight as a regulator on cell-cycle and macropinocytosis, we created lentivirus stable-transfected Cos7 and Skov3 cells with GRP75-knock-down (KD) and –over-expression (OE) of its phosphorylation mutants (Fig. 3C, D, Further file 2: Fig. S8). Circulation cytometry evaluation confirmed that GRP75-KD or phosphorylation-inactivation (T62A/S65A) considerably induced cell-cycle collected at G1-phase. In distinction, GRP75-OE or phosphorylation-activation (T62D/S65D) promoted cell-cycle S-phase enriched and M-phase arrested (Fig. 3E). Confocal checking the uptake of Tat/pGL3-Ca2+ nanoparticles confirmed that their macropinocytosis have been markedly diminished in GRP75-KD or phosphorylation-inactivation (T62A/S65A) cells, however considerably enhanced in GRP75-OE or phosphorylation-activation (T62D/S65D) cells (Fig. 4A, B). Such distinct uptake of Tat/pGL3-Ca2+ nanoparticles in GRP75-interrupted cells was additionally noticed in fluorescence quantification assays (Fig. 4C), and correspondingly resulted within the variation of transgene expression (Fig. 4D). Moreover, distinct macropinocytosis of Tat/pGL3-Ca2+ nanoparticles have been noticed in EGFP-positive, GRP75-transiently-transfection cells (Fig. 4E, F). These outcomes counsel that cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticle was pushed by high-expression or phosphorylation of GRP75.
Though additional work is required to deeply dissect why macropinocytosis was extremely upregulated by GRP75 and its phosphorylation (Fig. 4), the potential potentialities might be: (1) GRP75-OE enhance the expression of S-phase transcription issue E2F-1 and cyclin-dependent kinase inhibitor p21 . P21-activated kinase 1 (Pak-1) can promote CPPs uptake and macropinocytosis ; (2) GRP75 interacting with dynein gentle chain is concerned in membrane-associated trafficking . As a Pak1-interacting substrate, dynein gentle chain phosphorylation controls macropinocytosis . Subsequently, phosphorylation-modified GRP75 might considerably modulate this course of; (3) we have now demonstrated that GRP75-KD or inhibition considerably diminished Rac1 activation . Since Rac1 activation all the time induces membrane ruffling and macropinocytic cup formation , dynamic expression and phosphorylation of GRP75 from S- to M-phase modulate macropinocytosis is logical (Fig. 8E).
GRP75 promoted centrosome duplication by way of recruiting Mps1 to centrosome
Twin-specific protein kinase MPS1 controls various steps in cell-cycle. Its activation promotes centrosome duplication however inhibits mitotic checkpoint response . The discovering of co-enrichment of MPS1 with GRP75 throughout cell-cycle (Fig. 3A, B) promoted us to discover whether or not centrosome duplication was regulated by GRP75. Confocal checking the asynchronously rising Cos7 and Skov3 cells confirmed that GRP75 didn’t bind with unduplicated centrosomes throughout cell-cycle, however sure with partial fraction of duplicated centrosomes throughout cell-cycle (Fig. 5A-F, Further file 2: Fig. S9A-F). Subsequent, the affiliation of GRP75 with duplicated centrosomes from S- to M-phase in synchronously rising cells was noticed by microscopy examination (Fig. 5G–Ok, Further file 2: Fig. S9G-Ok). Since whole expression stage of GRP75 by the cell-cycle was at an analogous stage (Fig. 3B), phase-dependent affiliation of GRP75 with duplicated centrosomes promoted us to additional discover whether or not its expression or modification impacts centrosome duplication. In hydroxyurea (HU)-induced centrosome re-duplication assays (Fig. 5L–N, Further file 2: Fig. S9L-M), GRP75-OE considerably elevated the frequency of centrosome amplification, and GRP75-KD markedly diminished the centrosome amplification. Notably, extra elevated centrosome amplification was detected in GRP75 phosphorylation-activation (T62D/S65D) cells, whereas much less centrosome amplification was present in GRP75 phosphorylation-inactivation (T62A/S65A) cells (Fig. 5P, Further file 2: Fig. S9O). Related modifications of centrosome amplification have been present in asynchronously rising cells upon modulation of GRP75-expression stage (Fig. 5O, Further file 2: Fig. S9N). These outcomes counsel that high-expression or phosphorylated activation of GRP75 is required for centrosome duplication.
As chaperone protein complexed with mitotic kinases can modulate their exercise throughout the development of cell-cycle [57, 58], we subsequent examined whether or not centrosome-targeting of MPS1 was depending on the presence of GRP75. Confocal checking the affiliation of GRP75 and MPS1 at centrosome confirmed that these two targets have been unable to concurrently localize at centrosomes in GRP75-KD and phosphorylation-inactivation (T62A/S65A) cell. In distinction, vital centrosomal co-localization of GRP75 with MPS1 was detected in GRP75-OE and phosphorylation-activation (T62D/S65D) cells (Fig. 6A–F, Further file 2: Fig. S10A-F). Additional checking the centrosome-resident MPS1 in remoted fractions confirmed that GRP75-OE or phosphorylation-activation markedly elevated MPS1 stage, whereas GRP75-KD or phosphorylation-inactivation considerably diminished MPS1 stage at centrosome (Fig. 6G, H, Further file 2: Fig. S10G). These outcomes counsel that centrosome-recruited MPS1 was promoted by high-expression or phosphorylation of GRP75.
Moonlighting protein has unrelated features relying on the dynamic mobile context, which supplies a connection between distinct biochemical processes [52, 59]. We’ve got discovered that GRP75 is functionally enriched in heparan sulfate proteoglycan (HSPG)-mediated and membrane raft-associated endocytosis vesicles . It might probably moonlight as a cell-cycle controller and endocytosis regulator [24, 27, 28]. Right here, in OC cells, we additionally noticed that GRP75-depletion induced cell-cycle G1-phase accumulation and GRP75-over-expression induced M-phase enrichment (Fig. 3E), reinforcing its position on cell-cycle management. Earlier research reported that GRP75 may management cell-cycle progress by way of cyclin-dependent kinas/TP53/Rb signaling [54, 60]. Our current knowledge present that GRP75 related preferentially with duplicating centrosomes (Fig. 5, Further file 2: Fig.S9) and its phosphorylation was critically required for Mps1-translocating to centrosome (Fig. 6). Since MPS1 management centrosome duplication and mitotic checkpoint response and phosphorylated GRP75 super-activates Mps1 in a suggestions method [55, 56], the interdependence of GRP75 with MPS1 on centrosome-targeting (Fig. 6, Further file 2: Figs. S10, S11) means that GRP75 can be required for centrosome duplication and mitotic checkpoint response. Moreover, centrosome is a extremely unstable macromolecular complicated and Hsp70–Hsp90 chaperone equipment is important to take care of centrosome integrity for cell-cycle development [57, 58]. Thus, centrosome-recruiting of GRP75 from S- to M-phase (Figs. 5, 6) might effectively assist its maturation and correct meeting of the bipolar spindle.
GRP75-driven, cell-cycle-dependent macropinocytosis in nanoparticle remedy
To discover whether or not GRP75-driven, cell-cycle-dependent macropinocytosis contribute to the uptake of Tat/pDNA-Ca2+ nanoparticles in vivo, inhibitors of GRP75, cell-cycle and macropinocytosis have been utilized to modulate the supply of Tat/pDNA-Ca2+ nanoparticles in animals. NOD/SCID mice have been subcutaneously transplanted with Skov3 cells labeled with DF (double fusion, Fluc-eGFP) reporter gene, and Tat/TF (triple fusion, RFP-Rluc-HSV-ttk)-Ca2+ nanoparticles have been launched by tail vein injection for suicide gene remedy (Fig. 7A, B). Bioluminescence imaging confirmed that, with GCV administration to induce suicidal cell-killing, Fluc sign from all teams with Tat/TF-Ca2+ remedy elevated barely till d15, indicating environment friendly tumor-suppression by the nanoparticles. When macropinocytosis or GRP75 inhibitor was intratumoral injected, Fluc sign from EIPA and MKT077 teams considerably stronger than that of the PBS group (Fig. 7C, D). In parallel teams with intratumoral injection of cell-cycle inhibitors, Fluc sign from GCB- and PTX-groups sharply declined, indicating extra extensively regression of OC development than nanoparticle mono-therapy. In distinction, considerably greater Fluc sign from lovastatin- and Ro3306-groups have been noticed in comparison with the nanoparticle mono-therapy group (Fig. 7C, D). As a result of cell-cycle inhibitors distinctly affected OC regression by Tat/TF-Ca2+ nanoparticles, we tried moreover intratumoral injection with EIPA within the 4 take a look at teams. Evaluating to the elevated lovastatin- or Ro3306-induced Fluc sign, mixed therapy with EIPA additional markedly enhanced the Fluc sign, indicating decreased nanoparticle-therapy impact, as a result of diminished macropinocytosis at cell-cycle G1- or G2-phase. Notably, combining EIPA with GCB or PTX introduced reversed Fluc sign when in comparison with mono-treatment with GCB or PTX, indicating enhanced nanoparticle-therapy impact at S- or M-phase was on account of energetic macropinocytosis at these two cell-cycle sub-phases (Fig. 7C, D). Moreover, when MKT077 and EIPA have been mixed for intratumoral injection, the remedy impact of Tat/pDNA-Ca2+ nanoparticles was largely attenuated (Fig. 7C, D). We additionally checked physique weight of handled mice, and no vital modifications have been noticed in all experimental animals (Fig. 7E). TUNEL assays confirmed that therapy with Tat/TF-Ca2+ nanoparticle alone induced markedly apoptosis in OC tissues. Extra vital apoptosis was detected within the group with further GCB- and PTX-treatments, and elevated apoptosis was eradicated when EIPA was co-applied (Fig. 7F, Further file 2: Fig. S12). These knowledge strongly counsel that Tat/TF-Ca2+ nanoparticle-based suicide gene therapies have been dominantly managed by GRP75-driven, cell-cycle-dependent macropinocytosis.
To additional decide the position of GRP75-driven, cell-cycle-dependent macropinocytosis on supply of Tat/TF-Ca2+ nanoparticles, RLuc sign from TF plasmid was additionally investigated. As anticipated, Tat/TF-Ca2+ nanoparticles have been particularly collected at OC website (Fig. 8A, B). When EIPA or MKT077 have been additional launched, the OC-located RLuc sign from nanoparticles was sharply decreased, and was virtually diminished after the co-injection of the 2 inhibitors. Weaker OC-targeted RLuc sign was noticed after intratumoral co-injection with lovastatin or Ro3306, whereas additional co-injection with GCB or PTX introduced stronger RLuc sign at OC website (Fig. 8A, B). Once more, when EIPA was launched along with these cell-cycle inhibitors, synergistic TF RLuc sign was noticed in lovastatin- or Ro3306-injected mice, and reversed TF RLuc sign have been noticed in GCB- or PTX-injected mice (Fig. 8A, B). The contrasted concentrating on of Tat/TF-Ca2+ nanoparticles to OC and distinct tumor-regression impact brought on by completely different inhibitors was additional confirmed by ex vivo RFP imaging and histological staining of RFP expression on resected OC tissues (Fig. 8C, D). Noticeably, no visceral toxicity was noticed in OC-bearing mice handled by Tat/TF-Ca2+ nanoparticles with/with out aforementioned inhibitors (Further file 2: Fig. S13). Collectively, these outcomes counsel that GRP75-driven, cell-cycle-dependent macropinocytosis dominantly underlies Tat/TF-Ca2+ nanoparticle-based suicide gene remedy in OC (Fig. 8E).