Gunkel P , Iino H , Krull S , Cordes VC .

             nuclear pore complex assembly

	Figure 1. ZC3HC1 is a NE-associated protein in Xenopus laevis oocytes and cultured cells. (A) SEM micrographs of manually isolated and then differently treated X. laevis NEs from a weight class 1 frog’s late-stage V oocytes (for weight class definition, see Material and Methods). (A1) Nuclear and cytoplasmic face of NEs that had been isolated under NB-stabilising conditions (NB-s) preserving NPC and NB integrity while largely removing other NE-associated materials. (A2) Nuclear face of NEs treated with TX-100 and RNases, after having been isolated like the NEs presented in (A1), maintaining NB integrity. (A3) Nuclear and cytoplasmic face of the same NE, which had been treated with TX-100 but not with nucleases, following an NB-removal procedure that nonetheless allowed for maintaining the integrity of the lamina, the actual NPCs and the appendices at the NPCs’ cytoplasmic side. One of those sporadically observed NBs that had been disassembled only partially (arrowhead) is shown as a point of reference for the NE’s nuclear side. Bars, 100 nm; same magnification for all SE micrographs. (B) IB of subcellular fractions of X. laevis oocytes. (B1) IB of NEs that had been manually isolated from late-stage V oocytes, then incubated as two separate batches in parallel, either under NB integrity (+NB) or NB removal (-NB) conditions, subsequently extracted with TX-100, and finally sedimented by centrifugation. Labelling with indicated antibodies (target regions in parentheses) was on the upper and lower parts of the same membrane and on an identical duplicate, respectively. Since the total amount of loaded NE proteins did not exceed 500 ng per lane, resulting in polypeptide patterns not visible after staining by dyes like Ponceau S (PS), the stained membranes are not shown here. Note that ZC3HC1 was no longer present among the NE proteins after having applied conditions that removed most of the NB scaffold protein TPR and other NB proteins, like MAD1 and MAD2. By contrast, components of the NPC, like NUP62, had remained largely unaffected, and the NE-associated amount of NUP153 had only been slightly reduced in the absence of the NBs. (B2) IB of the soluble cytosolic proteins and particulate cytoplasmic materials (C) that after low-speed centrifugation had remained in the supernatant, the AL-containing membrane fraction, which still contained some contaminating yolk protein (asterisk marks major representative), the second wash of this latter fraction (W), and the total of proteins from the intact nuclei (N). All fractions stemmed from a batch of a weight class 3 frog’s stage VI oocytes that had been manually enucleated first. The oocytes’ other fractions, including other organelles and membranes, pigment granules, and the bulk of yolk, all of which were devoid of ZC3HC1 and TPR, are not shown here. Labelling for xlZC3HC1 and reference proteins xlTPR and xlNUP153, the latter recurrently detected also within the Xenopus oocyte’s AL fraction in trace amounts (blue arrowhead), and xlNUP62, as a regular part of the ALPCs, was on the upper and lower parts of the PS-stained membrane shown here, and on an identical duplicate. Each lane was loaded with the respective fraction or washing solution volume corresponding to only one oocyte, explaining why materials in lane N were hardly visible after PS-staining. Note that ZC3HC1, in stark contrast to NUP62, was not detectable in the AL fraction and instead turned out to be a primarily nuclear protein, just like TPR. (C) Triple-labelling IFM of cryostat sections of Xenopus oocytes with guinea pig, rabbit, and mouse antibodies for xlZC3HC1, xlTPR, and xlNUP62, respectively. Note that while NUP62 was seen both at the NE (arrowhead) and the cytoplasmic AL (some marked by arrows), ZC3HC1 and TPR were only detectable at the NE. The nucleus (N) and cytoplasmic compartment (C) of the stage VI oocyte are marked, as is the nucleus (asterisk) of an early-stage oocyte in which colocalisation at the NE occurred too. Oocytes were from a weight class 2 frog, with commonly less AL material than in class 3 frog oocytes. White dashed lines demark the cell boundaries of the early- and late-stage oocyte. Bar, 50 µm. (D) Double-labelling IFM of XL-177 cells for xlZC3HC1 and xlTPR, with the focal plane at the polar region of a representative nucleus. Note that both proteins at this resolution appeared to largely colocalise in dots, which for TPR were already known to represent NPC-associated NBs. Bar, 5 µm. (E) IFM of XL-177 cells for xlZC3HC1 and xlTPR and with a monoclonal antibody (mAb), mAb414, which binds to several NPC-located FG-repeat nucleoporins (NUPs). The focal plane was at the equator of a representative nucleus. Rectangles in the overlay micrographs were analysed by the ImageJ software, with line profiles plotted. Note the 4× enlarged line profile sections, showing almost complete overlap of IF-labelling for NE-associated ZC3HC1 and TPR, and an offset location of ZC3HC1 towards the nuclear interior relative to the FG-repeat NUPs, with approximate distances of around 150 nm between the line profile peaks for the labellings with mAb414 and for ZC3HC1. Note also the inverse order of labelling for ZC3HC1 and the FG-repeat NUPs at an NE invagination (arrowhead), compared to the NE’s not folded parts. Bar, 5 µm.
	Figure 2. ZC3HC1 is located at the NB primarily in the TR region. (A) Immuno-SEM of manually isolated X. laevis stage V oocyte NEs with antibodies for different parts of xlZC3HC1, the NT of xlTPR, and the NPBD of xlNUP153 and part of its NMBD. Representative composite images consist of SE micrographs of the NE’s nuclear face and the superimposed BSE images reflecting the IGPs’ positions, encircled in blue for NUP153, magenta for TPR, and green for ZC3HC1 (for the corresponding separate SE and BSE images, see Figure S3(D2)). Note that while the NUP153 antibodies decorated sites close to the NR, antibodies for ZC3HC1 primarily labelled the TR region, similar to IGP distribution for TPR’s NT. Bar, 50 nm. (B) Distributions of IGPs relative to an idealistic NPC and its NB in a face-on view. Schemes are drawn to scale, with the outer and inner diameters of the NR corresponding to 110 nm and 70 nm, and the outer diameter of the TR to 55 nm (for details regarding data acquisition and presentation, see Figure S3E,F). Bar, 50 nm. (C) Bar diagrams corresponding to the datasets presented in (B), representing the percentages of IGPs, grouped within windows of 5 nm width, that were located at differing distances away from the NB’s longitudinal axis, here represented by the y-axis. Values of the mean radial distances, provided together with standard deviation (SD) values, were deduced from the middle 68% of all measured distances values (contingents demarked by brackets) for those IGPs presented in (B) (for further information, see Figure S3(F1–6)). (D) Post-embedding iTEM on ultrathin sections of high-pressure-frozen, freeze-substituted, and then resin-embedded late-stage II oocytes of X. laevis. Sections had been labelled with antibodies for the central part of xlZC3HC1, the NT of xlTPR, and an N-terminal region of xlNUP153 that also comprises part of its TBD. Cytoplasm (C) and nuclear compartment (N), separated by the NE, are oriented toward the top and bottom. Parts of the NE segments presented, including their NPC wall-forming portions, are partially highlighted by white dashed lines to facilitate recognition. IGPs are shown encircled, with a diameter of 40 nm for the outer circles (for the rationale, see Figure S5C). Black arrows demark examples of NPCs in cross-section that had been IGP-decorated while the white arrow points at an NPC that had escaped labelling. White arrowheads point at parts of the NE where the actual phospholipid bilayer boundaries were not discernible, the black arrowhead at the example of an NPC whose non-diametric perpendicular section only yielded a small circle segment, and the double-headed arrows demark parts of the NE that appeared skewed and distorted for different reasons, with these and other features exclusion criteria (outlined in Figure S6A) for IGP distance measurements. Bar, 100 nm. (E) Bar diagrams representing the distribution of IGPs relative to the NE midplane (dashed vertical). Each histogram represents the mean of three different series of altogether several hundred measurements (see also Figure S6), comprising the percentages of those IGPs within windows of 10 nm width that were detected up to 100 nm and 200 nm away from the midplane of sectioned NPCs at their cytoplasmic (negative values) and nuclear sides (positive values). Provided values of mean distances and corresponding SD values are based on the middle 68% of all measured distance values for each target (contingents demarked by brackets). Note that ZC3HC1 and TPR-NT IGPs were found enriched in a similar region at the nuclear side of the NPC, which also harbours the NB’s TR, while the majority of NUP153-TBD IGPs were in closer proximity of the NE. (F) Incorporation of the three datasets, shown in (E), into one diagram as line graphs to facilitate comparison of relative IGP peak positions. Note that the major peak (numbered as 1) for ZC3HC1 was located slightly further away from the NPC midplane (dashed vertical) than the major one for the NT of TPR. Furthermore, in addition to each of these proteins’ major peaks, minor amounts of corresponding IGPs also appeared to be locally enriched, seemingly in intervals, further away from the NE, with the peaks for TPR-NT and ZC3HC1 marked by arrowheads in magenta and green, and the first additional minor peak for NUP153 by an asterisk. (G) Model for ZC3HC1 as a TR-resident and TPR-NT-neighbouring component of the NPC-attached prototypic NB, depicted in lateral view, and as a potential component of additional, sporadically observed TR-appended NB-like fibrillar structures (NBLS) described in Figure S7. Except for the fibril widths, the dimensions of the model segment meant to represent the NPC-attached NB, here numbered as ①, correspond approximately to those of the FA-fixed NB on an isolated Xenopus oocyte NE, commonly regarded as prototypic. In addition, this highly simplified model approximately outlines the contours of the NBLS, here numbered ②, ③ and +n, that can occur stacked on top of the TR of an NPC-attached NB, with these NBLS here depicted with increasing degree of transparency the further away positioned from the NB, to reflect the assumed decreasing frequency by which such NBLS occur stacked one on top of the other within the late-stage II oocyte. The length of such an NBLS is here depicted corresponding to about 42.5 nm. The green- and magenta-coloured clouds are meant to show the approximate distribution of those ZC3HC1 and TPR-NT IGPs that represent the major and minor peaks also arrowhead-marked in the respective colour in (F), with their positions relative to the NE midplane in (G) aligned to those in (F). Regarding the minor peaks’ coloured clouds, the increasing degree of transparency is approximately proportional to the corresponding peak decrease in (F). The width (here depicted as [a]) of the first clouds and their positioning relative to the NB’s central longitudinal axis is meant to reflect the relative positions of those IGPs detected in iSEM and shown in (B) that represent the central 68% of all signals around the mean distribution for TPR-NT and the mean for collectively all of the three ZC3HC1 datasets. The length (here depicted as [b]) of the first clouds and their positioning relative to the NPC’s midplane reflects the chord lengths of collinear sections, in parallel to the abscissa, through the main peaks for TPR-NT and ZC3HC1 in (F), which here corresponds to distances from the NPC midplane of 57–79 nm for TPR-NT and 69–98 nm for ZC3HC1. Bar, 50 nm.
	Figure 3. Widespread occurrence and anchorage of ZC3HC1 at the NEs of proliferating and non-dividing, terminally differentiated cells of different morphogenetic origin. (A) IFM of cryostat sections of the liver, the kidney’s renal pyramids and columns, and the forebrain’s prefrontal cortex from Macaca mulatta, representing tissues originating from the three germ layers. Overviews (left side) only show labelling for hsZC3HC1, while the micrographs at higher magnification also present the identical specimens’ triple-labelling with further antibodies, including such for TPR. For further comparison, liver and kidney sections were labelled with mAb414, while the cerebrum section was labelled for the neuronal cell marker RFOX3/NeuN to distinguish neuronal cells from glial cells. Note that ZC3HC1 and TPR were found colocalising at the NEs of cells present in these tissues. Bars, 100 µm (overview) and 10 µm, respectively. (B) IFM of non-dividing, mature X. laevis erythrocytes. The nuclei are shown in two focal planes, with the one on the nuclei’s equator and the other near the nuclei’s surface. Note the 4× enlarged line profile sections, showing ZC3HC1 at this level of resolution colocalising with TPR’s C-terminal domain, the latter known to be positioned in the TR region too. Bar, 10 µm. (C) IB of the LNN-enriched fraction of XL-177 cells and Xenopus erythrocytes obtained after extraction with TX-100, and of manually isolated and cleansed Xenopus oocyte NEs, obtained from a weight class 1 frog’s stage V oocytes and possessing intact NBs and NPCs, but hardly any of the different types of fibrillar appendices found appended in varying amounts to an oocyte NE from a weight class 3 frog. Loading amounts had been adjusted in such a manner that similar IB signal intensities were obtained for ZC3HC1. Labelling with indicated antibodies was performed on the upper and lower parts of the membrane stained with MemCode but here shown colour-converted from blue to red. Note that while the NE-associated amounts of TPR and ZC3HC1 relative to the reference NPC scaffold proteins NUP107 and NUP96 appeared higher in oocytes and XL-177 cells than in erythrocytes, the amount ratios between TPR and ZC3HC1 within the NB-enriched materials of these three different cell types appeared to be rather similar. (D) IB of cell extracts obtained from a confluent, not synchronised population of HeLa cells following fractionations of similar cell numbers performed in parallel, having applied either NB-s conditions, maintaining NB integrity, or NB-d conditions, causing partial or complete detachment of the one or other NB component from otherwise intact NPCs. Lanes were loaded with the non-fractionated cells’ total cell proteins (T), the soluble proteins released during extraction with TX-100 in NB-s or NB-d buffer (S), and the corresponding non-soluble pellet fractions (P). In addition, the same amounts of S and P materials from each fractionation were also loaded together in one lane to compare the resulting amount with that of the non-fractionated cells, demonstrating that hardly any amount of protein had been lost during the S and P fractions’ preparation. Labelling for hsZC3HC1, hsTPR, hsNUP153, and hsNUP107, and with mAb X223 cross-reactive with human LMNB2, the latter as an additional reference, was performed on the upper and lower parts of the MemCode-stained membrane shown colour-converted to red, and on an identical duplicate. Note that NUP153, anchored to the NPC’s NR, had remained bound to the LNN-enriched pelletable material, irrespective of whether NB-d or NB-s conditions had been applied, as it also was the case for NUP107 and LMNB2 (green arrowheads). Similarly, when having applied NB-s conditions, essentially all ZC3HC1 and TPR had remained bound to the LNN-enriched material too (arrowheads), even after the prolonged incubation at RT and despite efficient cell extraction and solubilisation of most of the cells’ other proteins. By striking contrast, essentially all ZC3HC1 had turned soluble (magenta-coloured large arrow) already after a short incubation at low temperature and in the absence of adequate amounts of divalent cations (for further details, see Materials and Methods). Further note that this had been accompanied by the loss of about half the cell’s total amount of TPR (magenta-coloured small arrow). As an aside, NUP107 polypeptides, also known to be part of ER-embedded individual pore complexes and small AL sheets, both of which are present in HeLa cells too (e.g., [99]), had contributed to the minor amounts of NUP107 in the soluble fractions following TX-100-extraction.
	Figure 4. Upon removal of phosphate modifications acquired during egg arrest at metaphase, soluble ZC3HC1 can re-engage in specific physical interactions with TPR even in solution. (A) IP of TPR and ZC3HC1 from 250,000× g supernatants of X. laevis egg extracts competent for post-mitotic nuclear assembly. IP of NUP62 from the same supernatant was performed as a control in parallel. Lanes were loaded with the total soluble cell proteins (T), with those proteins that had remained unbound (U) after bead incubation, those released during the third of three successive washing steps (W), and those obtained after final elution (E). Loadings in T and U represented the same volume fraction of the respective samples’ total amount (1 V), while the loadings in lanes W and E represented three-fold higher relative amounts (3 V). Immunolabelling was on the upper and lower parts of the PS-stained membrane shown here and on an identical duplicate. Cases in which a protein had been co-immunoprecipitated with the IP’s actual target protein are framed with brackets in green; brackets in magenta accentuate those in which no co-IP had occurred. Note that a subpopulation of TPR polypeptides had been co-immunoprecipitated together with ZC3HC1 and that, vice versa, a large proportion of the extract’s total content of ZC3HC1 had been co-immunoprecipitated with TPR. By contrast, neither TPR nor ZC3HC1 had been isolated together with NUP62, or vice versa. As an aside, note that some ZC3HC1 was found gradually detaching from the immunoprecipitated TPR during washes (blue arrowhead). Such polypeptides appear to represent one of seemingly two ZC3HC1 populations attracted by TPR, of which one is less tightly associated with TPR in vitro than the other. (B) IB of ZC3HC1 as the protein co-immunoprecipitated with TPR, as a component of manually isolated NEs from Xenopus stage V oocytes, and as a protein within the extract from metaphase-arrested Xenopus eggs, with such extract having been left untreated (t0), and after incubation for 15 min (t15) with λ phosphatase alone, and with the same amount of λ phosphatase supplemented with phosphatase inhibitor. The corresponding PS-stained membrane lacked the proteins below 30 kDa due to intentionally prolonged SDS-PAGE for better separation of ZC3HC1 polypeptides. Note that ZC3HC1 dephosphorylated with λ phosphatase exhibited electrophoretic mobility similar to that of the NB-associated ZC3HC1 polypeptides and those co-immunoprecipitated together with TPR from assembly-competent egg extracts.
	Figure 5. ZC3HC1 is strictly TPR-dependent for its own NE-association but also itself required for proper positioning of a substantial amount of TPR at the NB. (A) IFM of HeLa cells with antibodies for hsZC3HC1, hsTPR, and hsNUP153. Populations had been synchronized to increase the number of cells progressing through mitosis, next to some already in the G1 phase and a few still late in G2. Note that concurrent reassociation of ZC3HC1 and TPR with the NE (white arrows) was seen to occur late towards the end of telophase and early in G1, paralleling the onset of chromatin decondensation (yellow arrow), while NUP153 was part of the NE already notably earlier (white arrowheads). Bar, 10 µm. (B) RNAi experiments with HeLa and other cell lines, harvested at day 3 post-transfection. (B1) IB of whole-protein extracts from HeLa cells that had been transfected with control siRNAs (CTRL) or several different TPR and ZC3HC1 siRNAs (1–3). For estimating overall KD efficiencies, the total extracts from non-transfected cells had been loaded as serial dilutions (100 to 12.5%) in parallel, with the 100% loading equivalent to the amount from the same number of cells from the RNAi experiments. Immunolabelling was on different parts of the PS-stained membrane shown here. Note that TPR KD in this HeLa subline had resulted in a moderate reduction in the cellular amounts of ZC3HC1 (marked by arrowhead). (B2) IB of whole-protein extracts from U-2 OS and HCT116 cells transfected with control, TPR or ZC3HC1 siRNAs. Immunolabelling was on different parts of the shown membrane. Note that upon RNAi-mediated KD of TPR, cellular amounts of ZC3HC1 were reduced (arrowheads). (B3) IFM of HeLa cells treated with siRNAs. Upon TPR RNAi, only traces of TPR-staining were seen at most of the cells’ NEs; the usually bright NE-staining by TPR antibodies was only visible in cells that had remained non-transfected, here shown as a reference. Note that marked NE staining for ZC3HC1 was only seen in such TPR-positive cells (one marked by arrowheads), while in the TPR-deficient ones (one marked by white arrows), ZC3HC1 was found distributed throughout the nuclear interior. Upon ZC3HC1 RNAi, signal intensities for TPR at the NEs of the ZC3HC1-deficient cells (one marked by yellow arrows), compared to the non-transfected ones, appeared notably reduced, accompanied by the appearance of some additional staining for TPR within the nuclear interior, while NE-association of NUP153 did not appear affected. Further note that the micrographs for TPR are also shown colour-graded to display differences in pixel intensities via a colour look-up table (LUT), revealing a reduction of immunolabelling intensity for TPR at the ZC3HC1-deficient NE by about half. Bar, 10 µm.
	Figure 6. CRISPR/Cas9n-mediated ZC3HC1 gene disruption in HeLa cells neither prohibits cell cycle progression nor alters subcellular CCNB1 distribution. (A) IFM of HeLa WT cells grown together with cells of a stable HeLa ZC3HC1 KO line, with these mixed populations of WT and KO cells having been synchronised to the G1-phase. Two WT cells, positive for ZC3HC1, are marked by arrowheads. Note that in the neighbouring KO cells (examples marked by arrows), ZC3HC1 was neither detectable at the NEs nor anywhere else (see also Figure S15(B1)). Signal intensities for TPR at the NEs of the KO cells appeared reduced by about half, accompanied by the appearance of some additional staining for TPR within the nuclear interior, while NE-staining for NUP153 appeared unaffected. Bar, 10 µm. (B) IB of total cell extracts from HeLa WT and ZC3HC1 KO cells. Labelling with two different ZC3HC1 antibodies and for TPR, NUP153, and NUP107 for comparison was on the PS-stained membrane and on duplicates with identical loadings. A cross-reaction of one of the antibodies with an unrelated soluble protein is labelled by an asterisk. Note that ZC3HC1 was not detectable in this KO cell line and that TPR was only moderately reduced. (C) Time course of population growth of HeLa WT and ZC3HC1 KO cells. Data points with SDs were the mean results from three separate experiments, representing the growth of defined starting populations over 4 days. Note that the proliferation rate of the ZC3HC1 KO cells was similar to that of the WT progenitor cells. (D) IFM of co-cultured and cell cycle-synchronised HeLa WT and ZC3HC1 KO cells, harvested as a population enriched in cells in G2 and at the onset of mitosis. The cells were then immunolabelled for ZC3HC1 and CCNB1, and with an antibody targeting the phosphorylated serine 10 (S10p) of histone H3 (H3-S10p), as a very early hallmark for the onset of mitosis. Arrows mark cells at the G2/M transition point when staining for H3-S10p all along the NE is already prominent and when CCNB1 is readily imported into the nucleus within a time window of a few minutes. Arrowheads, by contrast, mark cells shortly before this time point. Note that while H3-S10 phosphorylation had already commenced in these arrowhead-marked cells, CCNB1 still appeared similarly well excluded from the nuclei of the WT and KO cells, even just before the actual end of G2. Bar, 10 µm. (E) IB of total cell extracts obtained from cell cycle-synchronised populations of WT and ZC3HC1 KO cells that had been harvested at indicated time points after the release from a thymidine-induced S-phase block. Immunolabelling for CCNB1 was performed on the PS-stained membranes shown here. Single asterisks mark BSA as part of trace amounts of culture medium not entirely removed from this experiment’s intentionally unwashed cells. Double asterisks mark a cross-reaction of the CCNB1 antibody, while CCNB1 itself, with a sequence-deduced Mr of about 48 kD, exhibited a gel-electrophoretic mobility equivalent to about 60 kD following SDS-PAGE by the current study’s commonly used method. Note that gradual CCNB1 accumulation over time, and its total amounts at corresponding time points, did not notably differ between WT and KO cells.
	Figure 7. Quantification of the relative amounts of NB-associated TPR in the WT and ZC3HC1 KO cells of four different cell lines reveals a reduction by about half upon the absence of ZC3HC1. (A) Quantification of the relative amounts of NB-associated TPR, in the HeLa WT and ZC3HC1 KO cells, following different procedures of specimen preparation and immunolabelling. (A1) IFM of mixed populations of HeLa WT and ZC3HC1 KO cells that had been co-cultured, cell cycle-synchronised, and harvested in G1. Cells were permeabilised with TX-100 either after (F > P) or before fixation (P > F), the latter resulting in the removal of the nuclear pool of soluble TPR. Labelling was with guinea pig antibodies for ZC3HC1, and a mAb for TPR, with the latter detected with either fluorophore-conjugated IgGs or a mouse IgG1-specific single-domain antibody (sdAb). The micrographs for TPR are again also shown colour-graded, displaying that signal intensity relationships between the WT and KO cells’ NEs were very similar within the different types of specimens. Bar, 10 µm. (A2) Quantification of signal yields for immunolabelled TPR at the NEs of HeLa WT and ZC3HC1 KO cells, following specimen preparation and immunolabelling as in (A1). Randomly chosen NE segments for quantifications via ImageJ were from essentially all labelled cells in equatorial view within randomly chosen images of mixed populations of WT and KO cells, with such images obtained from the four differently prepared specimens (for details, see Figure S16A). Box plots display the relative signal intensity values, with the arithmetic means marked by x, with the ones for the WT set to 100%, and with the SDs provided. Note that the mean TPR signal yield for the KO cells’ ZC3HC1-free NEs was only about half the WT cells’ corresponding value, largely irrespective of whether cells had been permeabilised before or after fixation or whether fluorescence stemmed from secondary IgGs or sdAbs. (B) Quantification of TPR signal yields at the NEs of WT and ZC3HC1 KO cells of HeLa, HCT116, U-2 OS, and hTCEpi. WT and ZC3HC1 KO cells co-cultured as mixed populations and harvested in G1 had been permeabilised with TX-100 after fixation, and immunolabelled for ZC3HC1 and TPR, with the latter then detected with fluorophore-conjugated, mouse IgG-specific sdAb. The dataset for HeLa represented an independent experiment distinct from the corresponding one presented in (A). Like for (A2), the mean TPR signal yield for the KO cells’ ZC3HC1-free NEs was only about half the WT cells’ corresponding value, with this applying to all the four cell lines. (C) IB of LNN materials obtained from WT cells of lines HeLa, HCT116, U-2 OS, and hTCEpi. Loading amounts had been adjusted for similarity of NUP107 signal intensities to facilitate comparability of the NE-associated amounts of TPR and ZC3HC1. Immunolabellings were on the membrane shown here and on a duplicate with identical loadings. The ZC3HC1-unrelated cross-reaction in the LNN materials of WT and KO cells of line hTCEpi, already addressed in Figure S15I, is marked by a double-asterisk. Note that amount relationships between TPR, NUP107, and ZC3HC1 were rather similar in the four different cell lines. As an aside, also note that the degree of post-translational modification of ZC3HC1 can differ between cell lines, with such modifications generally most pronounced in the LNN materials isolated from interphase and G0 populations of hTCEpi cells. (D) IB of LNN materials obtained from WT and ZC3HC1 KO cells of lines HeLa, HCT116, U-2 OS, and hTCEpi, harvested shortly before having reached confluency, in order to compare the relative amounts of NE-associated TPR in the ZC3HC1 KO versus WT cells for each cell type. Loadings for each cell line represented the same proportion of the WT and KO cells’ adjusted LNN materials (1 V), next to which half of this amount from the WT cells’ LNN fraction (0.5 V) was loaded as well. Immunolabelling was on the upper and lower parts of the membranes shown here and on identical duplicates. A double-asterisk again marks the ZC3HC1-unrelated cross-reaction in the LNN materials of hTCEpi. Note that for each cell line’s WT and KO cells, the signal intensity for NUP107 in the 1 V lanes, marked by arrow-tipped brackets in blue, was essentially the same. By contrast, TPR signal intensities in the LNN fractions of the ZC3HC1 KO cells amounted, at most, to only about half of the intensity in the corresponding WT cells’ fraction obtained from about the same number of cells. Accordingly, the signal intensities for TPR in the 1 V lanes of the KO cells of lines HeLa, HCT116, and hTCEpi were highly similar to those for TPR in the 0.5 V lanes of the corresponding WT cells, with such relationships marked by arrow-tipped horizontal brackets in dark green. In contrast, in the 1 V lane of the U-2 OS KO cells, the TPR signal intensity was slightly lower than in the WT cells’ 0.5 V lane, with this then marked by the double-headed curved arrow in lighter green.

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