EMBO/soda-nel huggingface dataset:
LETM1 couples mitochondrial DNA metabolism and nutrient preference (2018)
Figure 6

Figure 6. LETM1 haploinsufficiency in WHS is associated with abnormal mitochondrial morphology and ultrastructure.

(A) Steady state levels of LETM1 protein in fibroblasts from controls (C1-C3), WHS LETM1+/- (S1, S3, S4) and S5 the sole case of WHS in this study whose deletion does not encompass LETM1 (boxed in blue). GAPDH is shown as a loading control.

(B) Immunofluoresence analysis of control (C1), and WHS LETM1+/- S1-S4, fibroblasts with anti-TOM20 antibody. Scale bars are 8 µm in the main images and 3 µm in offset merged magnifications.

(C) Electron micrographs of controls (C1 and C2) and WHS LETM1+/+ (S5) cells; side panels show zooms. Scale bar: 0.2 µm. Black arrows indicate the areas shown offset at higher magnification.

(D) Electron micrographs of WHS LETM1+/- S1, S3 and S4 cells. Black arrows indicate elongated and branched mitochondria; side panels show magnifications. Scale bar: 0.2 µm.
LETM1 couples mitochondrial DNA metabolism and nutrient preference (2018)
Figure 8

Figure 8. WHS LETM1+/- fibroblasts show reduced PDH phosphorylation and tolerate a strict ketogenic diet.

(A) Representative immunoblots of mitochondrial lysates of HEK cells separated on a 20-42.5% iodixanol gradients probed for LETM1, PDH and TFAM.

(B) Left panel: immunoblot analysis of total PDH protein levels and the inactive phosphorylated (repressed form), PDHS293, in control (C1, C3), WHS LETM1+/- (S1, S3, S4) and WHS LETM1+/+ (S5) fibroblasts. Center and Right panels: Immunoblot analysis of PDK3 in control (C1-C3), WHS LETM1+/- (S1, S3 and S4) and WHS LETM1+/+ (S5) fibroblasts.

(C) Left panel: change in cell confluence of control (C1-C3), WHS LETM1+/- (S1-S4) and WHS LETM1+/+ (S5) fibroblasts grown in BHB over the course of 188 h. Data are expressed as means ± SEM of n=3 independent experiments. Right panel: images of control and WHS LETM1+/- S4 cells growing either in the presence of 25 mM glucose (HG) or BHB (KB) for 6 days. Scale bar 300 µm.

(D) Confocal images of control cells (C1) grown for 24 h in the presence of BHB and immunolabeled with anti-TOM20 (red) and anti-DNA (green), and DAPI stained (blue).

(E) Proportion of cells that displayed mtDNA aggregation during the KB treatment (panel i - control cells (C1, C2), and WHS LETM1+/+ (S5) fibroblasts; panel ii - LETM1+/- (S1, S2, S4) fibroblasts). At least 50 cells per cell line were counted at the indicated time points. Data are expressed as mean ± SEM of n=3 independent experiments.
Function of HNRNPC in breast cancer cells by controlling the dsRNA-induced interferon response (2018)
Figure 1

Figure 1. Knock-down of HNRNPC inhibited growth and tumorigenesis of breast cancer cells.

(A) Growth curves of the MCF7 (left) and T47D cells (right) upon gene silencing with siRNAs. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.

(B) Relative protein levels of HNRNPC upon siRNA-mediated silencing, quantified from Western blots of 3 replicates. The error bars represent the ± SD.

(C) Growth curves of the Tet-on Crispr-Cas9-MCF7 (left) and Tet-on Crispr-Cas9-T47D cells (right). Expression of Cas9 was induced with 5uM doxycycline after transfection of the sgRNAs. sgNC: non-targeting sgRNA as a negative control, sgHN-1: sgRNA sequence 1 for HNRNPC, sgHN-2: sgRNA sequence 2 for HNRNPC, sgLMNA: sgRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.

(D) Relative protein levels of HNRNPC upon CRISPR-mediated gene knock-down, quantified from Western blots of 3 replicates. The error bars represent the ± SD.

(E) Anchorage independent cell growth assays of the HNRNPC-deficient MCF7 (top) and T47D (bottom) cells.

(F) Relative protein levels of HNRNPC upon shRNA-mediated silencing, quantified from Western blots of 3 replicates. The error bars represent the ± SD.

(G) Images, weights, and growth records of the xenograft tumor models in female NSG mice established from the MCF7 cells with lentivirus-mediated stable gene knock-down. Each group has 6 mice. The error bars represent the ± SEM.

(H) Images, weights, and growth records of the MCF7-derived xenograft tumors, which were subjected to periodic siRNA injection, starting from 2 weeks after transplantation of the cells. Each group has 4 mice. The error bars represent the ± SEM.
Function of HNRNPC in breast cancer cells by controlling the dsRNA-induced interferon response (2018)
Figure 2

Figure 2. Repression of HNRNPC induced the interferon response and expression of ISGs in breast cancer cells.

(A, B) Volcano plots showing the differential expression of the genes after HNRNPC knock-down in the MCF7 (A) and T47D (B) cells. The ISGs with significant differential expression were marked as red dots. The cutoffs (dashed line) were set at the log2 fold-change > 1.5 or < -1.5 and the P-value < 0.001.

(C, D) Enrichment of the GO and KEGG functional annotations in the up-regulated gene sets upon HNRNPC knock-down in MCF7 (C) and T47D (D) cells. The P-values (-log10) of such enrichments were provided on the y-axis.

(E, F) qPCR measurements of the expressions of ISGs upon knock-down of HNRNPC in MCF7 and T47D cells using siRNA (E) or CRISPR-Cas9 (F). Each sample has 3 replicates. Error bars represent mean ± SD.

(G) 11 TFs involved in the interferon response and signaling, which were identified by MARINa as master TFs upon HNRNPC knock-down. In each row, all genes were sorted (from left to right) by their differential expressions in siHNRNPC vs. control cells. The predicted target genes that are positive or negative regulated by the TF are marked as red or blue bars. All the TFs were sorted by the P-values (FDR-corrected) from the MARINa analysis. Ranks of these 11 TFs among all the master TFs identified by MARINa were provided to the right.
Function of HNRNPC in breast cancer cells by controlling the dsRNA-induced interferon response (2018)
Figure 3

Figure 3. HNRNPC repression up-regulated the ISGs and suppressed MCF7 and T47D proliferation by activating the IFNβ production.

(A) Concentrations of IFNβ, measured by ELISA, in the culturing media of MCF7 and T47D cells 48 hours after siRNA transfections. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.

(B) The normal MCF7 (left) and T47D (right) cells were cultured in the media collected from the corresponding cells 48 hours after siRNA-mediated gene knock-down. Expressions of the ISGs in these normal cells were measured with qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.

(C) Different doses of the IFNβ antibody were added to the media of the siRNA-transfected MCF7 (left) or T47D (right) cells right before the media were transferred to the wild type cells. The wild type MCF7 or T47D cells were then cultured in these media for 48 hours, and the expressions of ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.

(D) Growth curves of the MCF7 (left) and T47D (right) cells cultured in the media collected from the siRNA-transfected cells. Each sample has 3 replicates. Error bars represent mean ± SD.
Function of HNRNPC in breast cancer cells by controlling the dsRNA-induced interferon response (2018)
Figure 4

Figure 4. Upregulated ISG expression and suppressed MCF7 and T47D proliferation upon HNRNPC repression mediated via the interferon beta signaling pathway.

(A-C) In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of IFNβ neutralization (A), IFNAR2 neutralization (B), or JAK-STAT inhibition (C). The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.

(D-E) Growth curves of the MCF7 (D) or T47D cells (E) upon HNRNPC knock-down but with the interferon response blocked with the IFNβ antibody. Each sample has 3 replicates. Error bars represent mean ± SD.

(F) Growth curves of the MCF7 (left) or T47D (right) cells upon HNRNPC knock-down and JAK-STAT inhibition with Ruxolitinib (5uM). Error bars represent mean ± SD. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. Each sample has 3 replicates. Error bars represent mean ± SD.
Function of HNRNPC in breast cancer cells by controlling the dsRNA-induced interferon response (2018)
Figure 5

Figure 5. The interferon response and tumor-inhibitory effect of HNRNPC repression were mediated via the dsRNA sensor RIG-I (DDX58).

(A, B) Expression levels of ISGs (A) and IFNβ concentration in the media (B) upon HNRNPC silencing in the cells with lentiviral shRNA-mediated stable knock-down of DDX58 or IFIH1. Each sample has 3 replicates. Error bars represent mean ± SD.

(C) Growth curves of the DDX58- and IFIH1-deficient MCF7 (left) and T47D (right) cells upon HNRNPC knock-down. Each sample has 3 replicates. Error bars represent mean ± SD.

(D) Xenograft tumor models with DDX58-deficient MCF7 cells. Images, weights, and growth records of the xenograft tumor models in female NSG mice established from the DDX58-deficient MCF7 cells upon lentivirus-mediated stable knock-down of LMNA or HNRNPC. Each group has 6 mice. The error bars represent the ± SEM.

(E) Xenograft tumor models with DDX58-deficient MCF7 cells. Images, weights, and growth records of the xenograft tumors derived from DDX58-deficient MCF7 cells, which were subjected to periodic siRNA injection, starting from 2 weeks after transplantation of the cells. Each group has 4 mice. The error bars represent the ± SEM.
Function of HNRNPC in breast cancer cells by controlling the dsRNA-induced interferon response (2018)
Figure 6

Figure 6. Repression of HNRNPC resulted in elevation of endogenous dsRNA.

(A) Immunofluorescence analysis of the dsRNA in MCF7 cells after knock-down of HNRNPC, with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) and anti-dsRNA antibody J2 (green). Cells transfected with poly I:C was included as a positive control of dsRNA, and the cells treated with RNase III was used as a negative control. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. The size of scale bar is 10um.

(B) Counts of dsRNA regions in the siNC control cells or in the cells with siHNRNPC, identified in the dsRNA-enriched libraries with anti-dsRNA J2 pull-down or in the control libraries including the IgG control and the input control for dsRNA pull-down. Each bar represents the overlapping dsRNA species from two replicate experiments. Among each set of the dsRNA regions, the dsRNAs from Alus were marked in grey.

(C) MA plot showing all the dsRNA regions identified in the dsRNA-enriched libraries. For each dsRNA region, mean of the read counts (log2) for two replicates of siHNRNPC and siNC cells was shown on the x-axis, and the average fold change (log2) in siHNRNPC vs. siNC on the y-axis. The up-regulated dsRNA regions were marked in red and down-regulated regions in black.

(D) An example of the up-regulated dsRNA regions upon HNRNPC knock-down. On top of the figure are plots of the read densities along the dsRNA region, in the dsRNA-enriched libraries (J2) and the two control libraries (IgG and input), from the siNC and siHNRNPC cells. This dsRNA region was originated from intron 4 of DDX21 transcript ENSG00000165732.12-001, which is shown at the bottom of the figure. The Alu DNA elements and the HNRNPC binding regions in this intron were also marked.

(E) From the results of RepeatMasker, percentage of the up-regulated dsRNA regions (928 regions highlighted in panel C) that contain or largely overlap with the Alu elements. For comparison, repeated for 10 times, the same number of intra-genic regions were randomly selected and the percentage of Alu was counted with RepeatMasker. The results, i.e., percentages of Alu in these 10 random sets, were summarized as the box plot.

(F) Percentage of the introns harboring the up-regulated dsRNA regions (698 introns in total, accounting for 864 of the 928 regions shown in panel C) that contain the previously identified HNRNPC binding regions. For comparison, 10 sets of randomly selected introns, which have the same length distributions as the 698 introns, were also queried. The results, i.e., percentages of the introns containing the HNRNPC binding regions in these 10 random sets, were summarized as the box plot.

Expanded View Figure Legends
Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control (2013)
Figure 1

Parkin promotes the biogenesis of cargo-selective MDVs in response to mitochondrial ROS

Schematic depicting the location of relevant protein markers relative to the mitochondrion, as well as the site of action of pharmacological agents used.
HeLa cells expressing GFP-parkin (green), OCT-DsRed2 (red) and CFP-Drp1K38E were left untreated (untreated) or treated for 2 h with 10 µM CCCP (CCCP), then fixed and stained for TOM20 (blue). Scale bars, 30 µm.
Cells prepared as in (B) were treated with 50 µM antimycin A for 2 h. Arrows indicate OCT-DsRed2-positive/TOM20-negative MDVs that colocalize with GFP-parkin, while circles indicate MDVs that are parkin-negative. Arrowheads indicate nascent vesicles, adjacent to mitochondria, exhibiting cargo selectivity and parkin recruitment. Open arrowheads indicate parkin-negative MDVs containing the reciprocal cargo (TOM20-positive/OCT-DsRed2-negative). Cell boundaries are delineated in the GFP-parkin single-channel image. Scale bars, 30 µm.
Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control (2013)
Figure 2

Wild-type parkin, but not PD-associated parkin mutants, promotes MDV biogenesis

Quantification of the number of OCT-DsRed2-positive/TOM20-negative vesicles in cells expressing GFP, GFP-parkinWT, GFP-parkinR42P, GFP-parkinK211N, or GFP-parkinC431F, treated with or without antimycin A (anti A) as in (C); both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P-values are given first for GFP-/GFP-parkin-positive vesicles, then for total vesicle number (n = 4968 cells in 23 experiments); ns, not significant; **P < 0.01; ***P < 0.001).
Structure of parkin, with PD-linked residues from (D) highlighted in dark gray (PDB ID: 4K95). Ubl, ubiquitin-like domain (red); RING0, RING0 domain (green); RING1, RING1 domain (cyan); IBR, in-between RING (magenta); REP, repressor element of parkin (yellow); RING2, RING2 domain (salmon).
HeLa cells expressing various GFP-parkin mutant contructs (green), pOCT-DsRed2 (red) and CFP-Drp1K38E were treated with 50 µM antimycin A for 2 h, then fixed and immunostained against TOM20. Arrows indicate matrix-positive/TOM20-negative structures colocalizing with GFP-parkin. Scale bars, 30 µm.
Immunoblot of whole-cell lysates of HeLa cells expressing various GFP-parkin constructs, treated with 50 µM antimycin A (anti A, +) or DMSO (-) for 2 h.

Source data are available online for this figure.
Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control (2013)
Figure 3

Parkin-dependent MDVs incorporate endogenous mitochondrial cargo

Representative immunoblot of whole-cell lysates from U2OS:GFP and U2OS:GFP-parkin cells transfected with non-targeting siRNA or siRNA targeting Drp1 (siDrp1).
U2OS:GFP-parkin cells transfected with siRNA targeting Drp1 were treated with DMSO (upper panels) or 25 µM antimycin A (anti A, lower panels) for 90 min, then fixed and immunostained against PDH (red) and TOM20 (blue). PDH E2/E3 bp-positive/TOM20-negative MDVs colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Scale bars, 20 µm (first panels on left) and 2 µm.
Quantification of PDH-positive/TOM20-negative vesicles in U2OS:GFP and U2OS:GFP-parkin cells transfected with the indicated siRNA, treated with DMSO or antimycin A (anti A) as in (B); both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P-values are given first for GFP-/GFP-parkin-positive vesicles, then for total vesicle number (n = 4885 cells in 23 experiments); ns, not significant; ***P < 0.001).
5 µm-long profile of the parkin-positive vesicle and adjacent mitochondrial tubule depicted in the antimycin A-treated cell from (B) (left). Increasing position values on the x-axis of the fluorescence intensity plot (right) correspond to moving from the top to the bottom of the profile. *MDV indicated by an arrow in (B).
Quantification of the distance between the centres of GFP-parkin-negative (black bar) or -positive (gray bar) vesicles and the edge of the nearest mitochondrial tubule for the vesicles quantified in antimycin A-treated U2OS:GFP-parkin cells transfected with siDrp1 in (C). Error bars represent the mean ± s.e.m.; *P < 0.05 (obtained by Student's t-test).
U2OS:GFP-parkin cells transfected with siRNA targeting Drp1 (siDrp1) were treated with 25 µM antimycin A for 90 min prior to fixation. Samples were immunostained against TOM20 (blue) and the indicated mitochondrial marker (red). TOM20-negative MDVs containing the specified cargo and colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Arrowheads show lack of colocalization between GFP-parkin and the indicated mitochondrial marker. Scale bars, 20 µm (first two columns of panels) and 2 µm (remaining columns of panels).
Quantification of TOM20-negative structures that stained positively for the indicated cargo in U2OS:GFP (GFP) and U2OS:GFP-parkin (GFP-parkin) cells, transfected with siRNA targeting Drp1, treated with antimycin A (anti A); both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P-values are given first for GFP-/GFP-parkin-positive vesicles, then for total vesicle number (n = 2459 cells in two experiments; ns, not significant; ***P < 0.001).

Source data are available online for this figure.
Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control (2013)
Figure 4

Parkin-dependent MDVs are targeted to lysosomes independently of autophagy

HeLa cells, transfected with siRNA targeting Drp1 and GFP-parkin (green), were treated with 25 µM antimycin A for 90 min following a 30-min pretreatment with 50 nM bafilomycin A1, then fixed and immunostained against PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp-positive/TOM20-negative MDVs colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Scale bar, 30 µm.
Quantification of PDH E2/E3bp-positive/TOM20-negative vesicles in HeLa cells treated with antimycin A in the presence of the lysosomal inhibitors bafilomycin A1 or pepstatin A and E-64d; both total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P-values are given for total vesicle number (n = 19 to 120 cells in 23 experiments; **P < 0.01; ***P < 0.001).
HeLa cells, transfected with GFP-parkin (green) and siRNA targeting Drp1 and Atg5, were treated with 25 µM antimycin A for 90 min, then fixed, immunostained for PDH E2/E3 bp (red) and TOM20 (blue), and counterstained for Hoescht (gray). PDH E2/E3 bp-positive/TOM20-negative MDVs colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Scale bars, 20 µm.
Quantification of PDH E2/E3 bp-positive/TOM20-negative vesicles from HeLa cells transfected with the indicated siRNA; both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P-values are given for the total vesicle number (n = 4264 cells in 23 experiments); ns, not significant.

Source data are available online for this figure.
Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control (2013)
Figure 5

PINK1 is required for parkin-dependent MDV formation

Representative immunoblot of whole-cell lysates from HeLa cells transfected with non-targeting siRNA or siRNA targeting PINK1 (siPINK1), treated with 10 µM CCCP for 6 h in order to stabilize the PINK1 full-length band.
HeLa cells transfected with GFP-parkin (green) and siRNA targeting PINK1 (siPINK1) or non-targeting control (ctrl siRNA), were treated with 25 µM antimycin A for 90 min, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp-positive/TOM20-negative MDVs colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Arrowheads indicate parkin-positive MDVs adjacent to mitochondria, possibly budding. Scale bar, 20 µm.
Quantification of PDH E2/E3 bp-positive/TOM20-negative structures from (B); both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P-values are given first for GFP-/GFP-parkin-positive vesicles, then for total vesicle number (n = 4260 cells in three experiments); ns, not significant; ***P < 0.001).

Source data are available online for this figure.
Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control (2013)
Figure 6

Parkin-dependent mitochondrial vesicle biogenesis is a rapid response triggered by ROS

Quantification of TMRM fluorescence by flow cytometry in U2OS:GFP-parkin cells treated with 25 µM antimycin A (anti A), 25 µM antimycin A and 10 µM oligomycin (anti A + oligo), and 20 µM CCCP (CCCP) for the indicated time, represented as a fraction of fluorescence intensity compared to DMSO-treated cells. Error bars represent the mean ± s.e.m. (n = 3 experiments).
U2OS:GFP-parkin cells were treated with DMSO (DMSO), 25 µM antimycin A (anti A), 25 µM antimycin A and 10 µM oligomycin (anti A + oligo), or 20 µM CCCP (CCCP) for two hours, then fixed and immunostained for TOM20 (red). Scale bars, 50 µm.
Quantification of the total number of PDH E2/E3 bp-positive/TOM20-negative structures in U2OS:GFP-parkin cells observed at various times for the indicated treatment. Error bars represent the mean ± s.e.m. (n = 46166 cells in 23 experiments).
U2OS:GFP-parkin cells were treated with DMSO (DMSO), 25 µM antimycin A (anti A), 25 µM antimycin A and 10 µM oligomycin (anti A + oligo), or 20 µM CCCP (CCCP) for two hours, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). A merged image of both mitochondrial markers (mito) is shown at the far right. Arrows indicate PDH E2/E3 bp-positive/TOM20-negative structures colocalizing with GFP-parkin (green). Scale bars, 20 µm.

Source data are available online for this figure.
Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control (2013)
Figure 7

Cell-wide clearance of mitochondria via autophagy is triggered by depolarization and proceeds over a 24-h time period

Representative immunoblot of whole-cell lysates from U2OS:GFP and GFP-parkin cells treated with DMSO, 25 µM antimycin A (anti A), 25 µM antimycin A with 10 µM oligomycin (anti A + oligo), or 20 µM CCCP for the indicated time period.
Quantification of PDH E1a signal intensity relative to that of actin in immunoblots from (A). Error bars represent the mean ± s.e.m. (n = 3 experiments).
Quantification of mitochondrial clearance in U2OS:GFP-parkin cells treated as in (A), fixed and immunostained for TOM20. Data are shown as percentage of cells containing mitochondria, by TOM20 staining, visualized by fluorescence microscopy. Error bars represent the mean ± s.e.m. (n = 3 experiments, with at least 85 cells quantified per condition, per experiment).
U2OS:GFP-parkin cells were treated as in (A) for the indicated time period, then fixed and immunostained for TOM20 (red). Cell boundaries are delineated in single-channel images. Scale bars, 20 µm.

Source data are available online for this figure.