EMBO/soda-nel huggingface dataset:
TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution (2020)
Figure 1

Figure 1. Depletion of TMEM135 results in cholesterol accumulation in lysosomes.

(A) RPE1 cells were transfected with either scramble or TMEM135 siRNAs and immunostained for LAMP1 as a lysosome marker (red) and PMP70 as a peroxisome marker (green). Scale bar, 10 μm.

(B) Quantification of overlap signal between lysosomes and peroxisomes shown in (A). Data represent mean ± SD (n=3 experiments), 50 cells were scored per condition per experiment. *P < 0.05, Student's t-test.

(C) Subcellular fractions of a discontinuous sucrose gradient collected from top to bottom, separated by SDS-PAGE, and immunoblotted for LAMP1, EEA1, Rab11, Rab8, PMP70, GM130, and Na+/K+ ATPase.

(D) Quantification of total cholesterol in the subcellular fractions shown in (C). Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.

(E) Cells transfected with TMEM135 siRNA were subjected to a SREBP2 cleavage assay for the precursor of SREBP2 and nuclear SREBP2.

(F) Cells transfected with TMEM135 siRNA were subjected to qPCR. Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.
TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution (2020)
Figure 2

Figure 2. Depletion of TMEM135 impairs ciliogenesis through disruption of intracellular cholesterol distribution.

(A) RPE1 cells were transfected with siRNAs as indicated, followed by serum starvation for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm.

(B) Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.

(C) Cells were transfected with siRNAs as indicated and incubated with HPβCD in serum-starvation media followed by filipin staining. Scale bar, 20 μm.

(D) Cells were transfected with siRNAs shown in (C) and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm.

(E) Quantification of the percentage of ciliated cells shown in (D). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.

(F) Cells were transfected with siRNAs as indicated, incubated with cholesterol-MβCD complex in serum-starvation media, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm.

(G) Quantification of the percentage of ciliated cells shown in (F). Data represent mean ± SD (n=3experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.
TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution (2020)
Figure 3

Figure 3. Depletion of TMEM135 impairs both trafficking and enhanced IFT20 recruitment to centrioles.

(A) RPE1 cells were transfected with siRNA as indicated, and immunostained for Rab8 (red) and γ-tubulin (green) in either 10% FBS medium (upper panel) or serum-starvation medium (lower panel). Scale bar, 10 μm. Arrowhead indicates Rab8 localized to the centrioles.

(B) Quantification of the percentage of cells with Rab8 localized to the centriole shown in (A). Data represent mean ± SD (n=3 experiments), 200 cells were scored per condition per experiment, *P < 0.05, Student's t-test.

(C) Cells were transfected with siRNA shown in (A) and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.

(D) Quantification of the percentage of IFT20 intensity at the centrioles in (c). Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.
TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution (2020)
Figure 4

Figure 4. Ectopic expression of a constitutively active form of Rab8 recovers ciliogenesis in TMEM135-depleted cells.

(A) RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild-type pGFP-Rab8a (WT-Rab8), constitutively active pGFP-Rab8a (Q67L) (CA-Rab8), or DN Rab8 dominant-negative pGFP Rab8a (T22N), incubated in serum-starved media for 12 h, and immunostained for ARL13B (Red), GFP-Rab8 (green), and DAPI (blue). Scale bar, 10 μm.

(B) Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD (n=3 experiments), 200 GFP-positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.

(C) (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST-JCF1 (RBD). The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. Bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.

(D) Cells were transfected as shown in (A) and cell lysates were incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected as shown in (A) and cell lysates were incubated with purified GST- protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody.
TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution (2020)
Figure 5

Figure 5. Malfunctioned ciliogenesis observed during TMEM135 depletion is not associated with IFT20.

(A) RPE1 cells were transfected with siRNAs as indicated, followed by 24 h incubation in serum-starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB.

(B) Efficiency of IFT20 knockdown by western blot.

(C) Cells were transfected with siRNAs as indicated, followed by incubation in serum-starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.

(D) Cells were transfected as shown in (C), and immunostained for Rab8 and γ-tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average (n=2 experiments).

(E) Cells were transfected with siRNAs as indicated, followed by transfection with Flag-IFT20, incubation in serum-starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag-IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm.

(F) Quantification of the percentage of ciliated cells with both the Flag-IFT20 and ARL13B localized in the cilium. Data represent mean ± SD (n=3 experiments), 150 Flag positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.

(G) Cells were transfected with siRNAs as indicated, followed by further transfection with CA-Rab8, incubation in serum-starvation media for 12 h, and immunostained for acetylated-tubulin. Representative fluorescent images of GFP-Rab8 Q67L (green), acetylated-tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm.

(H) Quantification of the percentage of GFP-positive ciliated cells (only those cilia having both GFP-Rab8 and acetylated-tubulin on cilium were considered for quantification). Data represent mean ± SD (n = 3 experiments), 200 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.
TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution (2020)
Figure 6

Figure 6. Enhanced IFT20 intensity induced by serum starvation at centrioles depends on Rab8 activation.

(A) Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells.

(B) RPE1 cells were transfected by siRNAs as indicated, followed by incubation in serum-starvation media for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm.

(C) Quantification of the percentage of ciliated cells shown in (B) Data represent mean ± SD (n = 3 experiments), 250 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.

(D) Cells were transfected by siRNa as indicated followed by incubation in serum-starvation media for 24 h, and immunostained for EHD1 (red) and γ-tubulin (green). Scale bar, 10 μm.

(E) Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in (D). Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.

(F) Cells were transfected by siRNAs as indicated, followed by incubation in serum-starvation media for 24 h, and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.

(G) Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in (F). Data represent mean ± SD (n=3 experiments), 150 were scored per condition per experiment, *P < 0.05, Student's t-test.

(H, I, J) Cells were transfected by siRNAs as indicated, followed by transfection with GFP-Rab8 WT, GFP-Rab8 Q67L, or GFP-Rab8 T22N, and further incubated in serum-starvation media for 12 h. Cell lysate was subjected to immunoprecipitation with anti-GFP antibody, followed by Western blot with antibody against GFP.
TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution (2020)
Figure 7

Figure 7. Cholesterol-MβCD complex rescues impaired Rab8 trafficking to the centriole, augments Rab8-GTP binding, and increases IFT20 intensity at the centriole in TMEM135-depleted cells.

(A) RPE1 cells were transfected as indicated, followed by incubation in serum-starvation media in the presence or absence of cholesterol/MCD for 24 h, and then immunostained for Rab8 (red) and γ-tubulin (green). Scale bar, 10 μm. Representative magnified images are shown from cells labeled with white asterisks.

(B) Quantification of the percentage of cells with Rab8 localized to the centriole shown in (A). Data represent mean ± SD (n=3 experiments), 200 cells were scored per condition per experiment, *P < 0.05, Student's t-test.

(C) (Upper panel) Cells were transfected as shown in (A) and cell lysate was incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) The intensity of bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. The bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.

(D) Cells were transfected as shown in (A), and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.

(E) Quantification of IFT20 intensity at the centriole shown in (D). Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.

(F) A proposed model showing the importance of cholesterol for primary ciliogenesis.

Expanded View Figure Legends
The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector (2022)
Figure 1

Figure 1. RipAC inhibits PTI in Arabidopsis

(A-C) RipAC overexpression inhibits ROS production induced by 50 nM flg22Pto (A), 100 nM elf18Rsol (B), and 200 μg/mL chitin (C) in Arabidopsis. ROS was measured as relative luminescence units (RLU) over time. Values are means ± SE (n=24 biological replicates).

(D) RipAC overexpression inhibits flg22-triggered MAPK activation in Arabidopsis. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points. Immunoblots were analysed using anti-pMAPK and anti-RipAC antibodies. Coomassie brilliant blue (CBB) staining and a non-specific band were used as loading control. Molecular weight (kDa) marker bands are indicated for reference.

(E) RipAC overexpression lines display elevated susceptibility to Pto DC3000 ΔhrcC, but not to Pto DC3000. Arabidopsis plants were spray-inoculated with indicated Pto strains (5x107 CFU mL-1) and bacterial titers were determined 3 days post-inoculation. Values are means ± SE (n=6 biological replicates). Asterisks indicate significant differences compared to Col-0 (Student's t test, ** p<0.01).

Data information: Experiments were performed 3 times with similar results.
The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector (2022)
Figure 2

Figure 2 RipAC associates with PUB4 in plant cells

(A-B) Interaction between RipAC and SlPUB4/AtPUB4 was confirmed by Split-LUC assay qualitatively (A) and quantitatively (B) in Nicotiana benthamiana. Values are means ± SE (n=8 biological replicates). Asterisks indicate significant differences compared to RipAC-nLUC/cLUC-AtPIP2A negative control (Student's t test, **** p<0.0001). RLU: relative luminescence units.

(C) Co-immunoprecipitation of SlPUB4/AtPUB4-GFP and RipAC-nLUC after co-expression in N. benthamiana. Two days after Agrobacterium infiltration, the plant tissues were collected and then subjected to anti-GFP immunoprecipitation. Immunoblots were analyzed using anti-LUC and anti-GFP antibodies. Molecular weight (kDa) marker bands are indicated for reference.

(D) Interaction between RipAC-RFP and SlPUB4/AtPUB4-GFP determined by FRET-FLIM upon transient co-expression in N. benthamiana leaves. GFP fluorescence lifetime of each GFP fusion protein is shown as a negative control. Lines represent average values (n=5 biological replicates) and error bars represent standard error. (Student's t-test, *** p<0.001).

Data information: Experiments were performed 3 times with similar results.
The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector (2022)
Figure 3

Figure 3. PUB4 positively regulates PTI in Arabidopsis

(A-D) ROS burst in Col-0 WT or the indicated pub4 mutant lines induced by 100 nM flg22Paer (A), 100 nM elf18Ecol (B), 1 μM AtPep1 (C) and 1 mg/mL chitin (D). ROS was measured as relative luminescence units (RLU) over time. Values are means ± SE (n=16 biological replicates).

(E) flg22-triggered MAPK activation in pub4 T-DNA mutants. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points for western blots. Immunoblots were analyzed using anti-pMAPK and anti-FLS2 antibodies. Coomassie brilliant blue (CBB) staining was used as loading control. Molecular weight (kDa) marker bands are indicated for reference.

(F-G) PUB4 contributes to seedling growth inhibition induced by 100 nM flg22Paer (F) or 100 nM elf18Ecol (G). Values represent the percentage of fresh weight of PAMP-treated vs water-treated seedlings, and are means ± SE (n=16 biological replicates). Asterisks indicate significant differences compared to Col-0 (one-way ANOVA, Kruskal-Wallis test, Dunn's multiple comparisons test, *** p<0.001, ****p<0.0001).

(H-J) PUB4 contributes to PAMP-induced stomatal closure. Stomatal apertures were measured as width to length ratio 2 h after treatment with mock or 10 μM flg22Paer (H), 1 mg/mL chitin (I) and 10 μM ABA (J). Values are mean ± SE (n>120). Asterisks indicate significant differences between samples (one-way ANOVA, Kruskal-Wallis test, Dunn's multiple comparisons test, ****p<0.0001, nsp>0.05).

(K) pub4 mutant lines display elevated susceptibility to Pto DC3000 COR- and Pto DC3000 ΔhrcC. Arabidopsis plants were spray-inoculated with indicated Pto strains and bacterial titers were determined 3 days post-inoculation. Values are means ± SE (n=6 biological replicates). Asterisks indicate significant differences compared to Col-0 (Student's t test, ** p<0.01).

Data information: Experiments were performed 3 times with similar results.
The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector (2022)
Figure 5

Figure 5. PUB4 associates with PRR complexes

(A) PUB4 associates in Arabidopsis with EFR, BAK1, and SERK2 after 1 μM elf18Ecol treatment, and constitutively with XLG2 and XLG1. PRR complex members were identified after immunoprecipitation of PUB4-FLAG from 35S:PUB4-FLAG line, tryptic digestion and sample analyses by LC-MS/MS. Untransformed Col-0 seedlings were used as a negative control. Total spectrum count for each protein is shown.

(B) Co-immunoprecipitation of PUB4 and BIK1 in N. benthamiana. PUB4-GFP or GFP-LTI6b were transiently co-expressed with BIK1-HA in the leaves of N. benthamiana. After treatment with mock or 1 μM flg22Paer for 10 min, total proteins (input) were extracted and subjected for immunoprecipitation with anti-GFP beads.

(C) Co-immunoprecipitation of PUB4 with PRR complex members in Arabidopsis. PUB4 co-immunoprecipitated with FLS2 and BAK1 specifically after 10 min of 1 μM flg22Paer treatment, and constantly with BIK1. Total protein extracts (input) from Col-0 and PUB4-FLAG plants were subjected to immunoprecipitation with anti-FLAG beads.

(D) PUB4 directly interacts with BIK1 in vitro. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference.

Data information: Experiments in (B-D) were performed 3 times with similar results.
The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector (2022)
Figure 6

Figure 6. RipAC does not affect PUB4 association with PRRs and BIK1.

RipAC does not affect PUB4 association with PRR complex members. PUB4-FLAG, PUB4-FLAG RipAC-GFP and Col-0 plants were treated for 10 min with water (as mock) or 1 μM flg22Paer and elf18Ecol treatment. Total protein extracts (input) were subjected for immunoprecipitation with anti-FLAG beads. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference. Data information: The experiment was performed 3 times with similar results.
The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector (2022)
Figure 7

Figure 7. PUB4 ubiquitinates non-active BIK1.

(A) PUB4 has auto-ubiquitination activity in vitro. PUB4 autoubiquitination assay using His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2) and MBP-PUB4 or MBP-PUB4 Cys239Ala inactive mutant reconstituted in bacteria. Ubiquitinated products were purified under denaturing conditions by immobilized metal ion chromatography (IMAC). Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates autoubiquitinated PUB4.

(B) PUB4 ubiquitinates non-active BIK1, and does not ubiquitinate active BIK1. Operon containing His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2), and MBP-PUB4 were co-transformed with GST, GST-BIK1, or GST-BIK1 kinase dead (BIK1-KD) in the presence of PUB4. Ubiquitinated products were purified under denaturing conditions by IMAC, and modified proteins further enriched by GSH chromatography. Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates the ubiquitination products.

Data information: The experiments were performed 3 times with similar results.
The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector (2022)
Figure 8

Figure 8. BIK1 accumulation is dually regulated by PUB4 and negatively affected by RipAC.

(A) Analysis of BIK1-HA protein accumulation in BIK1-HA+/-, pub4-/- BIK1-HA+/-, and PUB4-FLAG+/- BIK1-HA+/- in 4- to 5-week-old plants. Prior to protein extraction leave disks were treated for 6 h with 100 mM MG132, 50 mM CHX, and for 10 min with water (as mock) or 1 mM flg22Paer. Proteins were extracted in SDS buffer and analysed by western blot. BAK1 was used as a loading control. Numbers correspond to the quantitation of the BIK1-HA band, normalized to the quantitation of the BAK1 control band in the same sample.

(B) BIK1-HA accumulation is reduced in PUB4-FLAG+/- BIK1-HA+/- line compared to BIK1-HA+/- line in the absence of PAMP treatment. Total protein extracts were analysed by western blot. Tubulin was used as a loading control.

(C-D) PUB4 overexpression leads to reduction in ROS burst in response to (C) 100 nM flg22Paer and (D) 100 nM elf18Ecol. ROS was measured as relative luminescence units (RLU) over time. Values are means ± SE (n=24 biological replicates).

(E) BIK1-HA accumulation is reduced in RipAC-GFP/BIK1-HA line compared to Col-0/BIK1-HA line. Total protein extracts were analysed by western blot. Actin was used as a loading control.

Data information: In (A), (B), and (E), WB quantification was performed using ImageJ software. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference. Experiments were performed at least three times with similar results.
Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop (2021)
Figure 1

Figure 1. Plx1 binds to Apc1-loop500 via its Polo-box domain.

A. Schematic diagram of Apc1 is shown. Two loop domains (loop300, 294-399; loop500, 515-584) and PC repeat domain are shown in yellow and in cyan, respectively. Multiple alignment of sequences of Apc1-loop500 containing PBMs (Orange) and B56-binding site (green) are shown. Introduced alanine substitutions are indicated as A in red. Hs, Homo sapiens human; Pt, Pan troglodytes chimpanzee; Mm, Mus musculus mouse; Gg, Gallus gallus chicken; Xt, Xenopus tropicalis frog. Conserved or similar amino acids are shown with an asterisk (*) or dot (.), respectively.

B. Binding assay using MBP-fused Apc1-loop500 fragments. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, 2A (T532A/T539A) and S558A) was incubated with anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by immunoblotting with Plx1 antibody and Ponceau S staining.

C. Quantification of (B). The bar graph is quantification of bound Plx1. The intensities of Apc1-loop500 WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.

D. Specific binding of Plx1-PBD to Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives 2A (T532A/T539A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated in the presence of 10 µg of WT Plx1-PBD for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by Coomassie brilliant blue staining (CBB). Purified proteins (Plx1-PBD and Apc1-loop500) were run as a standard.

E. Quantification of (D). The bar graph is quantification of bound Plx1-PBD. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.

F. Plx1-PBD, but not its Pincer mutant, binds to Apc1-loop500. MBP-fused Apc1-loop500 WT was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated in the presence of indicated amounts of WT Plx1-PBD or Plx1-PBD Pincer mutant (Pin) for 15 min. The bound proteins were recovered by amylose beads and analysed as described in (D). The bound Plx1-PBD are shown as stoichiometries (the bound Plx1-PBD per Apc1-loop500) determined from Fig EV1.

G. CDK-dependent binding of Plx1-PBD to the Apc1-loop500. MBP-fused Apc1-loop500 was incubated in the presence or absence of purified Cdk2/cyclinA at 30˚C for 2 h. MBP-fused Apc1-loop500 (CDK+/-) was isolated by amylose beads and further incubated with indicated amounts of WT Plx1-PBD on ice for 10 min. The bound proteins were recovered by amylose beads and analysed as described in (D).