EMBO/soda-nel huggingface dataset:
Viral mediated redirection of NEMO/IKKγ to autophagosomes curtails the inflammatory cascade. (2012)
Figure 4

The C-terminal RNR R1 homology domain of M45 is required for the inhibition of NF-κB activation and interaction with NEMO.

(A) Schematic representation of M45 truncation mutants used in this and a previous [36] study. The C-terminal RNR R1 homology domain is shown in white, the unique N terminus in grey. The RHIM is marked black. (B) NIH-3T3 cells were transduced with retroviruses expressing full length M45, truncated M45 proteins, or GFP. After stimulation with IL-1β (20 ng/ml, 15 min), IκBα levels were determined by immunoblotting. (C) 293A cells were transfected with plasmids encoding Flag-tagged NEMO and HA-tagged full-length M45, truncated M45, or an unrelated MCMV control protein (m142), respectively. Lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody. Immunoprecipitates and the whole cell lysates (WCL) were analyzed by immunoblotting (IB) with the indicated antibodies. (D) Primary BMDMs were mock infected or infected with GFP-expressing wt MCMV (wt), ΔM45 mutant (ΔM45), M45 revertant (RM45), or MCMVs expressing Ct or Nt3 (RCt and RNt3) at an MOI of 1. 17 h postinfection cells were stimulated for 4 hours with TLR9 agonist CpG (0.5 µM) or TLR7 agonist R838 (0.1 µM) in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SEM).
Viral mediated redirection of NEMO/IKKγ to autophagosomes curtails the inflammatory cascade. (2012)
Figure 5

M45 induces lysosomal degradation of NEMO.

(A) NIH-3T3 cells were infected with MCMV ΔM45 or the revertant virus, RM45, at an MOI of 3. Cells were harvested at indicated time points postinfection, and the levels of IKK subunits, M45, IE1, and actin in lysates were analyzed by immunoblotting. (B) Murine fibroblasts (NIH-3T3, 10.1) or human embryonic kidney 293A cells were transduced with retroviral vectors expressing M45 or GFP, harvested 72 hours later, and analyzed by immunoblotting. (C) ts20 cells harboring a temperature-sensitive E1 enzyme were incubated for 14 h at a permissive (35°C) or a restrictive (40.5°C) temperature. Cells were then mock-infected or infected with RM45, and analyzed 10 h postinfection by immunoblotting (D) NIH-3T3 cells were mock infected or infected with RM45 (MOI of 5) and treated 2 hpi with 10 mM ammonium chloride (NH4Cl) or a mix of lysosomal protease inhibitors (PI-mix). 9 hpi, levels of IKK subunits, M45, IE1, and actin were determined by immunoblotting. (E) NIH-3T3 cells were stably transduced with retroviral vectors expressing M45 or GFP, and treated for the indicated times with PI-mix, 10 mM NH4Cl, or 5 µM lactacystin, respectively. Levels of the indicated proteins were analyzed by immunoblotting.
Viral mediated redirection of NEMO/IKKγ to autophagosomes curtails the inflammatory cascade. (2012)
Figure 6

M45 targets NEMO to autophagosomes.

(A) NIH-3T3 cells stably expressing Flag-tagged NEMO were mock infected or infected with wt MCMV-GFP or MCMV-GFP-ΔM45 at an MOI of 6. Cells were fixed 6 hpi, and NEMO distribution was analyzed by immunofluorescence. The arrow indicates the region which is shown in higher magnification in the right corner of the picture. (B) NIH-3T3 cells were transfected with Flag-NEMO and M45-HA expressing plasmids as indicated. 24 h later, cells were fixed and double immunofluorescence staining was performed using antibodies against the HA and Flag tags. Regions shown in higher magnification in the left corner of the pictures are indicated by arrows. (C) NIH-3T3 cells stably expressing Flag-NEMO were mock infected or infected with MCMV-M45-HA or MCMV-ΔM45 at an MOI of 7. Seven hpi, cells were fixed and analyzed as described for panel B. (D) NIH-3T3 cells were transfected with expression plasmids for Flag-NEMO and M45-HA or empty vector as indicated. Plasmids encoding GFP-tagged LC3, Rab5, or Rab7 were cotransfected. 24 hours posttransfection cells were fixed and used for anti-Flag immunofluorescence staining.
Viral mediated redirection of NEMO/IKKγ to autophagosomes curtails the inflammatory cascade. (2012)
Figure 7

Loss of NEMO degradation in autophagy-deficient MEFs, and MCMV-induced accumulation of LC3-II.

(A) atg5−/− and atg5+/+ MEFs were mock infected or infected with MCMVΔM45 or the revertant virus, RM45, at an MOI of 10. Eight hpi cells were harvested and levels of the indicated proteins were analyzed by immunoblotting. (B) 10.1 fibroblasts were mock infected or infected with wt MCMV or ΔM45 at an MOI of 5. Cells were left untreated or treated 30 min after infection with 10 mM NH4Cl to block lysosomal degradation of LC3-II. Cells were harvested at the indicated time points, and LC3-II levels in cell lysates were analyzed by immunoblotting using an LC3-II-specific antibody. LC3-II levels were quantified by densitometric analysis and normalized using the corresponding actin levels. Fold increases are shown relative to mock-infected cells.
Viral mediated redirection of NEMO/IKKγ to autophagosomes curtails the inflammatory cascade. (2012)
Figure 8

M45 induces the formation of autophagosomes and aggregates.

(A) NIH-3T3 cells stably expressing GFP-LC3 were transduced with retroviral vectors expressing M45 or Ct3. Three days later, cells were analyzed by confocal laser scanning microscopy. GFP-LC3 dots were counted in 50 cells per sample. Results are shown as box and whisker plots. The bottom and top of the box represent the first and third quartile, respectively. The horizontal line within the box represents the median. The minima and maxima within 1.5-fold interquartile range are shown as whiskers. Dots represent outliers. Significance was determined using ANOVA (***, p<0.001). M45 and Ct3 were detected by immunoblot. (B) Fluorescence images of cells described above. Large GFP-LC3 aggregates are indicated by arrow heads. (C) Electron micrograph of a double-membrane autophagosome in an M45-expressing cell. (D) Autophagosomes were counted in 10 randomly selected M45- and Ct3-expressing cells (2–3 TEM sections per cell). Results are shown in box and whisker plots as described for panel A. Significance was determined using the student's t-test (***, p<0.001). (E) Epifluorescence image of an M45-expressing cell with large GFP-LC3 structures. (F) TEM image of the same cell reveals large aggregates marked with ‘A’. (G, H) Successive enlargement of the region marked in panel F shows an example of an aggregate-associated autophagosome.
PTPN3 Acts as a Tumor Suppressor and Boosts TGF-β Signaling Independent of its Phosphatase Activity (2019)
Figure 2

Figure 2. PTPN3 promotes TGF-β signaling independent of its phosphatase activity.

A, Schematic diagram of PTPN3 domains. Deletion and point mutation of PTPN3 were shown.

B, Both PTPN3 and PTPN3 (L232R), but not the phosphatase-dead mutants PTPN3 (D811A) and PTPN3 (C842S), dephosphorylate P-EGFR (Y1173). Wildtype PTPN3 or its mutants were co-transfected with EGFR into HEK293T cells as indicated. 24 h later, cells were harvested for Western blotting. Levels of P-EGFR (Y1173), EGFR, PTPN3 and its mutants were examined by appropriate antibodies.

C, PTPN3 and PTPN3 (D811A) enhance TGF-β-induced target protein expression. HaCaT cells stably expressing HA-PTPN3 or HA-PTPN3 (D811A) were stimulated with 2 ng/mL of TGF-β for 6 h and then harvested for cell lysates. Protein levels were examined by Western blotting with appropriate antibodies as indicated.

D, PTPN3 and its catalytically inactive mutants PTPN3 (D811A) and PTPN3 (C842S) equally enhance TGF-β-induced reporter gene activity. HaCaT cells were transfected with expression plasmids for each PTPN3 variant and CAGA-luc reporter and Renilla-luc reporter. Cells were harvested for relative luciferase assay after TGF-β (2 ng/mL, 8 h) treatment. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.

E, PTPN3 FERM domain enhances TGF-β-induced reporter gene activity as potently as wildtype PTPN3. HaCaT cells were transfected with expression plasmids for PTPN3 truncations and CAGA-luc reporter, and internal control Renilla-luc reporter. Cells were harvested for relative luciferase assay with TGF-β treatment (2 ng/mL, 8 h). Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. **P<0.01, ***P<0.001. NS, non-significant.
PTPN3 Acts as a Tumor Suppressor and Boosts TGF-β Signaling Independent of its Phosphatase Activity (2019)
Figure 3

Figure 3. PTPN3 attenuates ubiquitination of TβRI to stabilize TβRI

A, PTPN3 and PTPN3 (D811A) stabilize TβRI. TβRI and PTPN3 were detected by Western blotting with appropriate antibodies as indicated in HaCaT cells stably expressing PTPN3 or PTPN3 (D811A).

B, shRNA PTPN3-mediated reduction of endogenous TβRI level is restored by MG132. HaCaT cells expressing shRNA PTPN3-1, shRNA PTPN3-2 or control shRNA were treated with MG132 (20 μM) for 4 h. Cell lysates were analyzed by Western blotting with anti-TβRI, anti-PTPN3 and anti-GAPDH antibodies.

C, PTPN3 rescues the degradation of TβRI induced by Smurf2. HEK293T cells were transfected with the indicated plasmids. Cells were harvested after 24 h post-transfection. Western blotting was done with the indicated antibodies.

D, PTPN3 and PTPN3 (D811A) attenuate ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids. 24 h after transfection, IP and immunoblotting analysis were performed with the anti-FLAG antibody. Levels of TβRI ubiquitination and other proteins were examined by Western blotting with the indicated antibodies.

E, PTPN3 blocks Smurf2-mediated ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids, and treated with MG132 (20 μM, 4 h) before harvest. TβRI IP was performed with the anti-HA antibody, and TβRI ubiquitination was determined by Western blotting with the anti-Myc antibody. Protein levels were examined by Western blotting with the indicated antibodies.

F, Depletion of Smurf2 reverses the shPTPN3-induced degradation of TβRI. HEK293T cells were transfected with expression plasmids for shPTPN3, shSmurf2 and HA-TβRI. 48 h post-transfection, cells were harvested for immunoblotting analysis. TβRI level was determined by Western blotting with the anti-HA antibody. Levels of other proteins were done by Western blotting with the indicated antibodies.

G, Dominant negative Smurf2 mutant C716A prevents shPTPN3-induced TβRI degradation. HaCaT cells stably expressing shPTPN3 were transfected with FLAG-Smurf2 (C716A). Cells were harvested for Western blotting with anti-TβRI, anti-FLAG, anti-PTPN3 and anti-GAPDH antibodies.
PTPN3 Acts as a Tumor Suppressor and Boosts TGF-β Signaling Independent of its Phosphatase Activity (2019)
Figure 4

Figure 4. PTPN3 attenuates the binding the Smad7-Smurf2 E3 ligase to TβRI

A, PTPN3 interacts with TβRI at endogenous levels. HaCaT cells were harvested after TGF-β treatment (2 ng/mL, 1 h) and then subjected to IP by PTPN3 antibody or control IgG. PTPN3 and TβRI were detected by Western blotting.

B, Both PTPN3 and PTPN3 (D811A) interact with TβRI. FLAG-TβRI was co-transfected with HA-PTPN3 or HA-PTPN3 (D811A) into HEK293T cells. Cell lysates were harvested and subjected to IP with FLAG antibody. PTPN3, PTPN3 (D811A) and TβRI were detected by Western blotting with appropriate antibodies.

C, The FERM domain of PTPN3 is necessary for its binding to TβRI. FLAG-TβRI and HA-PTPN3 (wildtype or truncations) were co-transfected into HEK293T cells. Cells were harvested after 24 h and subjected to IP by HA antibody. TβRI, PTPN3 and PTPN3 truncations were detected by Western blotting with appropriate antibodies.

D, Schematic representation of TβRI deletion mutants and their interactions with PTPN3 or Smad7. EC, extracellular domain; TM, transmembrane domain; GS, Gly-Ser-rich motif; +, positive interaction; -, no interaction.

E, Knockdown of endogenous PTPN3 promotes the Smad7-TβRI association. HEK293T cells were transfected with the indicated plasmids and treated with MG132 (20 µM) for 4 h before harvested. SFB-Smad7 proteins were retrieved with the Streptavidin pulldown. Levels of TβRI and other proteins were examined by Western blotting with the indicated antibodies.

F, PTPN3 and PTPN3 (D811A) abolish the interaction between TβRI and Smurf2 in HEK293T cells. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 μM) for 4 h, followed by IP and Western blotting.
PTPN3 Acts as a Tumor Suppressor and Boosts TGF-β Signaling Independent of its Phosphatase Activity (2019)
Figure 5

Figure 5. PTPN3 (L232R) fails to augment TGF-β signaling.

A, PTPN3 wildtype, phosphatase-dead D811A and ICC-derived mutant L232R exhibit similar patterns of subcellular localization and co-localization with TβRI. HaCaT cells were transfected with HA-TβRI and Flag-PTPN3 (wildtype or its mutants). 24 h later, cells were harvested for immunofluorescence with the indicated antibodies. Scale bar = 10 μm.

B, PTPN3 (L232R) does not increase the TβRI protein level. TβRI and PTPN3 were determined by Western blotting in HaCaT cells stably expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R).

C, PTPN3 (L232R) fails to rescue the Smurf2-mediated degradation of TβRI. HEK293T cells were transfected with FLAG- TβRI, Myc-Smurf2 and the increasing anount of PTPN3 (wildtype and mutants). Cells were harvested 24 h post-transfection. Protein levels were examined by Western blotting with the indicated antibodies.

D, PTPN3 (L232R) does not block the interaction between TβRI and Smurf2. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 μM) for 4 h and analyzed by IP-Western blotting.

E, Ectopic expression of PTPN3 wildtype and PTPN3 (D811A), but not PTPN3 (L232R), completely abolishes the Smad7-TβRI interaction. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 µM) for 4 h, followed by IP and Western blotting.

F, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), attenuate ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids. 24 h after transfection, IP-Western blotting analysis was performed with anti-Flag antibody IP and followed by Western blotting with the indicated antibodies.
PTPN3 Acts as a Tumor Suppressor and Boosts TGF-β Signaling Independent of its Phosphatase Activity (2019)
Figure 6

Figure 6. PTPN3 enhances TGF-β-induced growth inhibitory responses

A, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), enhance Smad2 and Smad3 phosphorylation. HaCaT cells were stimulated with TGF-β (2 ng/mL) for 1 h. Levels of P-Smad2, P-Smad3, Smad2, Smad3, PTPN3 and GAPDH were detected by Western blotting.

B, PTPN3 (L232R) inhibits TGF-β-induced reporter gene activity. HepG2 cells were transfected with expression plasmids for PTPN3, PTPN3 mutants and CAGA-luc reporter, and Renilla-luc reporter. Cells were harvested for luciferase assay after 8 h of TGF-β (2 ng/mL) treatment. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. *P<0.05, **P<0.01.

C, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), enhance TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by MTS assay. Data are shown as mean ± SEM; n = 3.

D, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing shPTPN3 were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by using MTS assay. Data are shown as mean ± SEM; n = 3.

E, PTPN3 (L232R) attenuates TGF-β-induced growth inhibition on colony formation in HepG2 cells. Cells were treated with or without TGF-β (1 ng/mL) treatment for 10 days. Clonogenic assay is described in Materials and Methods. Quantification of the results in clonogenic assays was analyzed by Image J. Data are shown as mean ± SEM; n = 3.

F, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect in HepG2 cells. Cells with stable knockdown of PTPN3 were treated with or without TGF-β (1 ng/mL) for 10 days. Clonogenic assay is described in the main text. Quantification of the results in clonogenic assays were analyzed by Image J. Knockdown efficiency of PTPN3 in HepG2 cells were analyzed by Western blotting with the anti-PTPN3 antibody. Data are shown as mean ± SEM; n = 3.

G, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect in Huh7 cells. Experiments and analysis were similarly done as in (F). Data are shown as mean ± SEM; n = 3.
PTPN3 Acts as a Tumor Suppressor and Boosts TGF-β Signaling Independent of its Phosphatase Activity (2019)
Figure 7

Figure 7. PTPN3 enhances TGF-β-induced growth inhibitory responses

A, PTPN3, PTPN3 (D811A) and PTPN3 (L232R) are similarly expressed in Huh7 stable cell lines. Expression levels of indicated proteins were detected with appropriate antibodies in Western blotting.

B, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), attenuates tumorigenesis. Luciferase-harboring Huh7 tumor cells (2 × 106 cells/mouse) expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) or empty vector (served as negative control) were subcutaneously injected into 5-week-old nude mice. Twenty days after injection, mice were analyzed by bioluminescence using Xenogen IVIS imaging system. Tumors expressing luciferase is indicated by radiance bar (p/sec/cm2/sr).

C, Quantitation of bioluminescence with data from (B). Data are shown as mean ± SEM; n = 5.

D, Depletion of PTPN3 accelerates tumorigenesis. HepG2 cells stably expressing shPTPN3 or shControl were subcutaneously injected into nude mice. Fifty days after cell implantation, tumors were dissected and photographed.

E, Depletion of PTPN3 increased tumor sizes. Tumor volume was measured at indicated days post-implantation. Data are shown as mean ± SEM; n = 6 for each group.

F, Depletion of PTPN3 increased tumor weight. Weight from all tumors was recorded from both control and shPTPN3 groups. Data are shown as mean ± SEM; n = 6 for each group.

G, PTPN3 depletion impairs TGF-β signaling in tumors. Levels of PTPN3, GAPDH and TGF-β signaling associated proteins, such as TβRI, P-Smad2, P-Smad3, Smad2/3, Smad4, PAI-1, c-Myc and p15 in tumors were analyzed by Western blotting analysis.

H, Clinical significance of PTPN3 expression in LIHC based on TCGA database. In the boxplots, Y-axis represents the expression level of PTPN3 in normal and tumor samples. The boxes show the median (horizontal line in the box) ± 1 quartile; the upper and lower box limits represent the upper and lower quartiles, respectively. The whiskers (vertical lines) extend from the hinge to the smallest or larget value within 1.5 interquartile range from the box boundaries.
Genome-wide RNAi screen reveals a role for multipass membrane proteins in endosome-to-golgi retrieval. (2014)
Figure 3

Characterization of SFT2D2 Function in Endosome-to-Golgi Retrieval

(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and SFT2D2 KD cells (bottom).

(B) Cells stably expressing SFT2D2-Myc show localization of SFT2D2 to perinuclear membranes and endosomes, including VPS35-positive endosomes (arrowheads in inset). Treatment with nocodazole disperses the endosomes and even more clearly shows colocalization of SFT2D2-Myc and VPS35 (arrowheads in inset).

(C) Cells stably expressing SFT2D2-Myc were costained with antibodies against various post-Golgi SNARE proteins. Colocalization was quantified by Pearson’s correlation coefficient (right-hand graph) and indicates very extensive colocalization with STX6, STX7, and VAMP8. The image panels illustrate colocalization of SFT2D2-Myc and STX5 at the Golgi (top, arrowhead) but much more extensive colocalization of SFT2D2-Myc and STX6 (bottom, arrowheads). In (B) and (C), the white dashed box delineates the area magnified in the insets.

(D) SNARE protein staining was compared for control and SFT2D2 KD HeLa cells and quantified. The graph shows the change in cellular intensity measured for each post-Golgi SNARE investigated. Images illustrate the increased cellular intensity of VAMP3 in SFT2D2 KD cells.

(E) Control and SFT2D2 KD HeLa cell lysates were separated by LDS-PAGE and blotted for the indicated SNARE proteins or actin. Scale bars in (A)–(D), 20 μm, except insets in (B), 1 μm, and in (C), 2 μm. Quantitation in (C) and (D) was done using automated microscopy (see Experimental Procedures); error bars indicate SD of two separate multicell experiments. Average Pearson’s correlation coefficients measured were often identical in repeated experiments.
Genome-wide RNAi screen reveals a role for multipass membrane proteins in endosome-to-golgi retrieval. (2014)
Figure 4

Localization and Characterization of ZDHHC5 in HeLa Cells

(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and ZDHHC5 KD cells (bottom).

(B) Cells stably expressing ZDHHC5-Myc show localization of ZDHHC5 to the plasma membrane and to intracellular tubules and vesicles. Some colocalization between ZDHHC5 and retromer VPS35 is observed (arrowheads in inset). Following nocodazole treatment endosomes are dispersed and some are labeled with ZDHHC5 and VPS35 (arrowheads in inset).

(C) Control and ZDHHC5 siRNA-treated SFT2D2-Myc cells were mixed and stained for ZDHHC5, Myc, and VPS35. KD cells are marked by an asterisk.

(D) Control and ZDHHC5 siRNA-treated HeLa cells were mixed and stained for ZDHHC5, α5-integrin, and VPS35. KD cells are indicated with an asterisk.

(E) Control (top) and ZDHHC5 KD (bottom) HeLa cells were fixed and stained for TGN46 and β1-integrin.

Scale bars in (A)–(E), 20 μm, except inset in (B), 2 μm.
Genome-wide RNAi screen reveals a role for multipass membrane proteins in endosome-to-golgi retrieval. (2014)
Figure 5

GRINA Knockdown or Overexpression Inhibits Endosome-to-Golgi Retrieval

(A) Primary screen anti-CD8 antibody uptake images for control cells (top) and GRINA KD cells (bottom).

(B) Control (top) and GRINA KD (bottom) HeLa cells were stained for Golgi marker GM130 and Golgi glycoprotein-1 (GLG1).

(C) Cells stably expressing GFP-Rab6 were treated with GRINA siRNA (bottom) and compared to control cells (top) upon staining for TGN46 and VPS35.

(D) Quantitation by western blotting of lysates from control and GRINA-silenced HeLa cells and GFP-Rab6 cells.

(E–G) HeLa cells were transiently transfected with GRINA-Myc for 24 hr before fixing and staining. (E and F) GRINA-Myc colocalizes with both TGN46 and VPS35. In this example, GRINA-Myc expression (in the cell marked by an asterisk in E) reduced the cell’s TGN46 expression compared to surrounding untransfected cells and caused enlargement of VPS35-positive endosomes. The area in (E) magnified in (F) is indicated by a dashed line. (G) GRINA-Myc expression also perturbs CIMPR localization. In this example, GRINA-Myc transfection (in the cell marked by an asterisk) caused CIMPR to localize to round vesicular structures positive for GRINA-Myc, some of which appeared larger than regular endosomes, whereas TGN46 staining was almost absent in the transfected cell.

Scale bars in (A)–(C), (E), and (G), 20 μm, and in (F), 10 μm.