EMBO/soda-nel huggingface dataset:
Dephospho-CoA kinase, a nuclear-encoded apicoplast protein, remains active and essential after Plasmodium falciparum apicoplast disruption (2021)
Figure 3

Figure 3 E. coli DPCK complementation in the apicoplast and the deletion of DPCK

A.) Construct used to express the E. coli DPCK in the apicoplast. Appended are the N-terminal signal sequence and transit peptide from ACP (api) and a C-terminal mCherry tag.

B.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue).

C.) Genotyping PCR confirming the deletion of the endogenous DPCK, with amplification demonstrating integration at the Δ5' and Δ3' loci and lack of wild type parasites (as indicated by the failure to amplify at the wild type 5' and 3' loci) as compared to the parental control. A schematic describing these genotyping PCR reactions is shown in Figure EV3.

D.) Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck parasite line that is expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue).

E.) Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck line after treatment with 1x azithromycin for 7 days under supplementation with 50μM mevalonate. This parasite line expresses api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue).

F.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We failed to amplify sufB from the azithromycin-treated (+ Azith) PfMev api-EcDPCK-mCherry Δdpck parasites, indicating the loss of the apicoplast organellar genome.

Data information: All microscopy images represent fields that are 10µm long by 10µm wide. Colocalization statistics are shown in Table 1.
Dephospho-CoA kinase, a nuclear-encoded apicoplast protein, remains active and essential after Plasmodium falciparum apicoplast disruption (2021)
Figure 4

Figure 4 E. coli DPCK complementation in the cytosol and deletion of DPCK

A.) Construct used to express the E. coli DPCK protein with a C-terminal mCherry tag in the cytosol of the PfMev parasite line.

B.) Live epifluorescence microscopy of the PfMev EcDPCK-mCherry line. This parasite line expresses api-SFG (green) and EcDPCK-mCherry (red) with DAPI marking the nuclei (blue).

C.) Genotyping PCR confirming the deletion of the endogenous DPCK using primer pairs described in Figure EV3.

D.) Live epifluorescence microscopy of the PfMev EcDPCK-mCherry Δdpck line. This parasite line expresses api-SFG (green) and EcDPCK-mCherry (red) with DAPI marking the nuclei (blue).

E.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We were successful in amplifying sufB from the PfMev EcDPCK-mCherry Δdpck parasite line grown under continuous supplementation with 50μM mevalonate, indicating retention of the apicoplast organellar genome.

Data information: All microscopy images represent fields that are 10µm long by 10µm wide. Colocalization statistics are shown in Table 1.
Dephospho-CoA kinase, a nuclear-encoded apicoplast protein, remains active and essential after Plasmodium falciparum apicoplast disruption (2021)
Figure 5

Figure 5 Characterization of the PfMev CLD-EcDPCK-mCherry-apt Δdpck line under permissive and non-permissive conditions

A.) Construct used to generate the PfMev CLD-EcDPCK-mCherry-apt parasite line.

B.) Genotyping PCR confirming the deletion of the endogenous DPCK (Δdpck) using primer pairs described in Figure EV3.

C.) Live epifluorescence microscopy of the PfMev CLD-EcDPCK-mCherry-apt Δdpck line. This parasite line expresses api-SFG (green) and CLD-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). This parasite line was grown in the presence of 0.5μM aTc.

D.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the absence of aTc for 48 hours. The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite.

E.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the presence of 0.5μM aTc, and in the presence of 0.5μM Shield1 (Shld) for 48 hours.

F.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the absence of aTc, and in the presence of 0.5μM Shield1 (Shld) for 48 hours. The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite.

G.) Growth curve of the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown either in the presence of 0.5μM aTc and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM aTc and the presence of 0.5μM Shield1 (non-permissive condition, red). Pantothenate was supplied at 50nM in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 1 (two-way ANOVA, followed by Bonferroni's correction; *, P < 0.05). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate.

Data information: All microscopy images represent fields that are 10µm long by 10µm wide. Colocalization statistics are shown in Table 1.
Dephospho-CoA kinase, a nuclear-encoded apicoplast protein, remains active and essential after Plasmodium falciparum apicoplast disruption (2021)
Figure 6

Figure 6 Characterization of azithromycin-treated PfMev CLD-EcDPCK-mCherry-apt Δdpck parasites under permissive and non-permissive conditions

A.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We were unsuccessful in amplifying sufB from the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line after treatment with 100nM azithromycin (Azith) in the presence of 50μM mevalonate, indicating the disruption of the apicoplast organelle.

B.) Live epifluorescence microscopy of the apicoplast-negative PfMev CLD-EcDPCK-mCherry-apt Δdpck line post treatment with 100nM azithromycin in the presence of 50μM mevalonate. This parasite line expresses api-SFG (green) and the CLD-EcDPCK-mCherry construct (red) with DAPI marking the nuclei (blue). Images represent fields that are 10µm long by 10µm wide.

C.) Growth curve of the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line containing a disrupted apicoplast grown either in the presence of 0.5μM aTc and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM aTc and the presence of 0.5μM Shield1 (non-permissive condition, red). Pantothenate (50nM) and mevalonate (50μM) were supplied in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 2 (two-way ANOVA, followed by Bonferroni's correction; **, P < 0.01). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate.

D.) Upon apicoplast disruption, DPCK is located within vesicles that contain apicoplast-specific proteins and may still be able to generate CoA.
Placensin is a glucogenic hormone secreted by human placenta (2020)
Figure 2

Figure 2 - High expression of FBN2 and placensin in human, but not mouse, placenta.

A FBN2 mRNA levels in multiple human tissues. Human tissue cDNAs were used as templates for PCR of FBN2, followed by agarose gel analyses. GAPDH levels served as loading controls (biological replicates, n=2).

B FBN2 mRNA levels in mouse tissues based on quantitative RT-PCR. Data were normalized based on levels in adipose tissues.

C Comparison of FBN2 transcript levels between human and mouse placentas. Common primers for FBN2 and GAPDH in both species were used for RT-PCR analyses (biological replicates, n=3) .

D Immuno-histochemical detection of placensin in human placental villi obtained from women at 9 and 38 weeks of pregnancy. During early pregnancy, placensin immunostaining was found in cytotrophoblasts (c), whereas syncytiotrophoblasts (s) showed weak staining. At 38 weeks of pregnancy, strong placensin signals were detected in both syncytiotrophoblasts and cytotrophoblasts (arrows). Staining using E-cadherin antibodies confirmed the identity of cytotrophoblasts (Inset showing higher magnification E-cadherin (E-Cad) staining). Lower panels depict sections stained with nonimmune IgG as negative controls. Scale bars, 100 μm.

E Immunoblotting of placensin in human placental villi (term pregnancy). Samples from three individual patients were processed with or without N-glycosidase pre-treatment to reveal untreated and deglycosylated placensin.

F For prokaryotic cell expression, a cDNA fragment corresponding to residues 2779 to 2912 of the human FBN2 coding region was appended with GST and 6-histidine tags and sub-cloned into the PGEX-6P-1 vector for expression in E. coli. Left panel: Coomassie blue staining of proteins in the SDS-PAGE gel before and after digestion with the PreScission protease. 1: Flow through; 2: Washes; 3: Before PreScission treatment; 4: After PreScission treatment. Right panel: Immunoblotting using placensin antibodies.

G Immunoblotting of recombinant placensin generated in E. coli. and CHO cells. For CHO cell expression, placensin cDNA sequence was subcloned into an adenoviral vector pAV[Exp]-CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later. Recombinant placensin proteins secreted from CHO cells (b) showed higher molecular weight than those from bacteria (a). Following treatment with N-glycosidase to remove N-linked carbohydrate side chains, prokaryotic- and eukaryotic cell-derived placensin proteins showed similar sizes.

Data information: Bars are shown as mean±SEM. a,b: groups with different letters were significantly different (p<0.05) by parametric unpaired Welch's t test.
Placensin is a glucogenic hormone secreted by human placenta (2020)
Figure 3

Figure 3 - Placensin stimulation of cAMP production, PKA activity, and glucose release in hepatocytes and promotion of glucose secretion in vivo.

A Prokaryotic cell-derived placensin stimulated cAMP production (at 10 min.), and PKA activity (at 30 min.) by primary mouse hepatocytes and hepatocyte-derived AML12 cells (biological replicates, n=5).

B Placensin stimulation of glucose secretion (at 5h) and key gluconeogenesis gene transcripts (at 1h) in both cell types (biological replicates, n=4). Transcript levels for PEPCK and G6Pase were determined using quantitative RT-PCR. Treatment with glucagon (10 nM) served as positive controls.

C Liver from adult female mice was perfused in situ with placensin (1.0 ug/pulse) or PBS before measurement of hepatic glucose output from inferior vena cava (biological replicates, n=3).

D Adult female mice were injected intraperitoneally with placensin (30 ug/injection) with blood sampling at different time points for glucose levels (biological replicates, n=5).

Data information: Bars are shown as mean±SEM. For A and B, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by ANOVA followed by Brown-Forsythe and Welch's test; For C, *p<0.05, ****p<0.0001, significant differences between placensin injection groups and corresponding control groups by parametric unpaired Welch's t test; For D, **p<0.01, significant differences between placensin injection groups and corresponding control groups by nonparametric Mann-Whitney t test; *p<0.05 and ****p<0.0001, significant differences between control groups and placensin injection groups by parametric unpaired Welch's t test.
Placensin is a glucogenic hormone secreted by human placenta (2020)
Figure 4

Figure 4 - Placensin secretion and promotion of trophoblastic cell cAMP production, PKA activity, MMP9 expression, and invasion.

A Left panel: Immunoblotting of placensin content in HTR-8/SVneo, but not BeWo, JAR, and JEG3, cells. Right panel: Time-dependent secretion of placensin by HTR-8/SVneo, but not JAR, cells monitored using an ELISA (biological replicates, n=4-5).

B Knockdown of FBN2 in HTR-8/SVneo cells led to decreases in FBN2 transcripts and placensin secretion. Cells were treated with FBN2 siRNA (SiFBN2; 20 nM) or control siRNA (SiC; 20 nM) for 6h. Following media change, cells and conditioned media were collected at 48h after incubation. Placensin transcripts and proteins were determined by RT-PCR and ELISA (biological replicates, n=8), respectively. Lower panel: immunoblotting of secreted placensin by HTR-8/Svneo.

C Placensin stimulation of cAMP production and PKA activities by HTR-8/SVneo cells. Cells were treated with placensin for 10 min. before cAMP measurement (biological replicates, n=5). For PKA activities, cells were treated with placensin (50 ng/ml) for 30 min. Treatment with forskolin (10 uM) served as positive controls.

D Placensin stimulation of transcript levels for MMP9 but not MMP2(biological replicates, n=12). Cells were treated with placensin for 24h before RT-PCR analyses of transcript levels.

E Placensin stimulation of MMP9, but not MMP2, activities without affecting cell proliferation. After 24h of treatment with placensin, media were collected for zymographic and densitometric analyses as well as determination of total cell numbers (biological replicates, n=4).

F Placensin stimulation of HTR-8/SVneo cell invasion. After placensin treatment for 24h, cell invasiveness was determined using the Transwell assay (left panel: micrographs of cells; right panel: fold changes in number of migrated cells, biological replicates, n=4). Cells were incubated with mitomycin C to rule out effects on cell proliferation. Scale bars: 100μm. C: control.

Data information: Bars are shown as mean±SEM. For B, ANOVA followed by Tukey's multiple comparison tests was used for comparison. For C, ANOVA followed by Brown-Forsythe and Welch's test was used for comparison. For D/E/F, parametric unpaired Welch's t test was used. When comparing with control groups, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
Placensin is a glucogenic hormone secreted by human placenta (2020)
Figure 5

Figure 5 - Increases in serum placensin and minimal changes in glucagon levels during human pregnancy and in patients with gestational diabetes mellitus (GDM).

A Immunoblotting of serum placensin from patients at second trimester (1-3), term pregnancy (4-6) using specific placensin antibodies.

B Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients. Numbers in parentheses represent number of patients.

C Ages of patients at different gestational stages and BMI values for normal pregnant women and GDM patients.

Data information: Bars are shown as mean±SEM. *p<0.05, nonparametric Mann-Whitney's t test was used for comparison between GDM groups with corresponding control groups.

Expanded View figures
N-glycan processing selects ERAD-resistant misfolded proteins for ER-phagy (2021)
Figure 1

Fig. 1. N-glycan processing and delivery of ATZNNN to endolysosomes.

(A) N-glycan. Glucoses, blue; mannoses, green; N-acetylglucosamines, black. GI removes glucose 1. GII removes glucoses 2 and 3. UGGT1 adds-back glucose 3. UGGT1 can only modify the terminal mannose on branch A (black circle). αMI and EDEM1, EDEM2 and EDEM3 remove light green, α1,2-bonded, mannose residues. CST inhibits GI and GII. KIF inhibits α1,2mannosidases.

(B) AATNNN and ATZNNN. N-glycans in red. The E342K mutation of ATZNNN is in blue.

(C) Control of polymerization propensity for the Halo-tagged versions of AAT (lanes 1 and 4) and of ATZ (lanes 2 and 5). Polymers and monomers corresponding to (Miranda et al., 2010; Ronzoni et al., 2021) are seen with anti-Halo (lanes 1-3) or with the polymer-specific 2C1 antibody (lanes 4-6). Synthesis control in SDS-polyacrylamide gel by visualization of tetramethylrhodamine (TMR) ligand-labeled Halo-AATNNN and Halo-ATZNNN (lanes 7-9).

(D) HaloTag pulse-chase analysis in WT MEF monitoring fluorescent Halo-AATNNN during up to 6 h of chase in the presence of BafA1. The fluorescent JF646-Halo ligand and the polymer-specific 2C1 antibody stains are shown.

(E) Same as (D) for fluorescent Halo-ATZNNN.

(F) Quantification of accumulation of fluorescent Halo-AATNNN and Halo-ATZNNN within LAMP1-positive endolysosomes. Mean ± SEM, n=20, 11, 10, 13 for Halo-AATNNN, n= 18, 12, 12, 13 for Halo-ATZNNN. Two-way ANOVA and Sidak's multiple comparison test, ns P>0.05, *** P<0.001.

Data information: Scale bars 10 µm.
N-glycan processing selects ERAD-resistant misfolded proteins for ER-phagy (2021)
Figure 2

Fig. 2. Role of CNX and of N-glycan processing in delivery of ATZNNN to the endolysosomes.

A Confocal laser scanning microscopy analyses in cells treated with BafA1. Total HA-ATZNNN (red) or HA-ATZNNN polymers (magenta) in LAMP1-positive endolysosome (green) in WT MEF.

(B) Radiolabeled ATZNNN enters in 2C1-positive polymers that are immunoisolated at the end of the chase times. BafA1 inhibits polymers degradation. Quantification for n=4. Unpaired two-tailed t-test, **** P<0.0001.

(C) Same as (A) in Cnx-KO MEF.

(D) Same as (A) in WT MEF exposed to kifunensine (KIF).

(E) Same as (A) in WT MEF exposed to Castanospermine (CST).

(F) Same as (A) in sCNX MEF.

(G) Same as (A) in Uggt1-KO MEF.

(H, I) (H) Quantification of HA-positive or (I) of 2C1-positive endolysosomes (mean, n= 18, 14, 17, 13, 12, 12 cells for panels A, C-G , respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001.

(J) Halo-ATZNNN in Uggt1-KO MEF mock-transfected (empty pcDNA3 plasmid).

(K, L) (K) Same as (J), with a plasmid for expression of active or (L) inactive UGGT1.

(M) Quantification of Halo-ATZNNN-positive endolysosomes (mean, n=22, 10, 15 cells for panels J-L, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001).

Data information: Scale bars 10 μm.
N-glycan processing selects ERAD-resistant misfolded proteins for ER-phagy (2021)
Figure 3

Fig. 3. N-glycosylation at position 83 is required and sufficient for ATZ delivery to endolysosomes.

(A) ATZ glycosylation mutants and their electrophoretic mobility. N is the acceptor site asparagine; Q is for the mutation to glutamine that prevents glycosylation. The E342K mutation of ATZNNN is in blue.

(B-I) Confocal laser scanning microscopy analyses (as in Fig. 2) in WT MEF treated with BafA1.

(J, K) (J) Quantification of ATZxxx-positive endolysosomes and (K) of ATZxxx polymers-positive endolysosomes (mean, n= 18, 30, 24, 14, 27, 22, 27, 18 cells for HA stain and n= 12, 14, 14, 11, 14, 14, 17, 11 cells for 2C1 stain of panels B-I, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001.

Data information: Scale bars 10 μm.
N-glycan processing selects ERAD-resistant misfolded proteins for ER-phagy (2021)
Figure 4

Fig. 4. Intracellular fate of ERLAD-competent vs. ERLAD-incompetent ATZXXX glycosylation variants.

(A) Radiolabeled ATZXXX enter in 2C1-positive polymers that are immunoisolated at the end of the given chase times. Total ATZNNN is immunoisolated with HA. Protein load and 35S-methionine/cysteine incorporated in nascent polypeptides are shown.

(B) Quantification of radiolabeled polymers. Green columns are for the ERLAD-competent ATZNNN and ATZQNQ variants (see Fig. 3B, 3G, 3J, 3K), red columns are for the ERLAD-incompetent ATZQQN and ATZQQQ (see Fig. 3H, 3I, 3J, 3K) (mean, n=5 for ATZNNN, n=3 for ATZQNQ, n=4 ATZQQN and n=4 for ATZQQQ). Two-way ANOVA and Sidak's multiple comparison test, ns P>0.05, * P<0.005, ** P<0.01, *** P<0.001, **** P<0.0001.

(C) Co-immunoprecipitation of ATZXXX (Bait, lower panel) with CNX (upper panel) and BiP (middle panel). Protein content in the total cell extracts (TCE) is shown.

(D) Quantification of (C) for CNX association (n=3). Unpaired two-tailed t-test, ** P<0.01*** P<0.001, **** P<0.0001.

(E) Quantification of (C) for BiP association (n=3 for ATZNNN and ATZQQN; n=4 for ATZQNQ and ATZQQQ). Unpaired two-tailed t-test, * P<0.005, ** P<0.01, *** P<0.001, **** P<0.0001.

(F) Detergent-insoluble fraction of (C).

(G) Confocal laser scanning microscopy in Flp-In 3T3 cells stably expressing ATZNNN in the absence of BafA1 (to allow clearance of misfolded model proteins delivered within endolysosomes). Co-localization with Calnexin (CNX).

(H) Same as (G) for BiP co-localization.

(I) Same as (G) for ATZQQQ.

(J) Same as (H) for ATZQQQ.

(K) HaloTag pulse-chase analysis in MEF monitoring fluorescent Halo-ATZNNN during up to 24 h.

(L) Same as (K) for fluorescent Halo-ATZQQQ. Arrowheads show select clusters of JF646-labeled Halo-ATZQQQ. Arrowheads show co-localization Halo-ATZQQQ:BiP.

Data information: Scale bars 10 μm.
Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity (2011)
figf1

(a,b) Multiple MBds associate with a PC3 cell (a) and a B-lymphoblast (b). Insets: MBd labelling (a) and merged differential interference contrast microscopy image with MBd labelling to show cell boundaries (b). MKLP1, MBd marker (a,b; red); CD44, membrane (a; green); DAPI, DNA (a; blue). Scale bar, 5 μm (a) and 2 μm (b). (c,d) Three-dimensional reconstructions of polarized cells in a monolayer (c) and a HeLa cell (d) show intracellular MBds. (c) ZO-1, tight junction; MKLP1, MBds. Scale bar, 2 μm. Enlargement (bottom panel) of the box in the top panel shows five MBds (arrows). (d) Wheat-germ agglutinin, plasma membrane (red); MKLP1–GFP, MBds (green); DAPI, DNA (blue). Scale bar, 5 μm. (e) Electron micrograph of an MBd in a permeabilized MCF-7 cell showing immungold labelling with MKLP1 antibodies. Inset, lower magnification of the MBd (boxed) in the cell; nucleus, right. Scale bar, 200 nm. (f) Time-lapse images during extracellular trypsin treatment of HeLa cells show retention of most MBds (MKLP1–GFP, red). Two MBds (yellow arrows) are lost on treatment, indicating digestion and/or dissociation. Time (h:min) post trypsin. Scale bar, 5 μm. (g) Two-day co-cultures of HeLa cells expressing either MKLP1–GFP (MBd marker) or cytosolic RFP. Green MBds (arrows) associated with red cells (asterisk) indicate post-mitotic transfer of MBds between cells. Scale bar, 10 μm.
Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity (2011)
figf2

(a) The CETN1–GFP signal is brighter in the upper centrosome/spindle pole of a mitotic spindle. The merged differential interference contrast microscopy image with CETN1–GFP labelling at two centrosomes shows a metaphase chromosome. Insets: enlargements and semi-quantitative integrated intensity profiles of centrioles. Scale bar, 5 μm. (b) The brighter CETN1–GFP signal represents the older centrosome, as it co-stains more intensely for hCenexin1 and remains more intense throughout cell division (Supplementary Fig. S1a). Scale bar, 5 μm. Lower left, merge. (c,d) Time-lapse images show that the mitotic midbody is preferentially inherited by the daughter cell with the older centrosome in HeLa cells (c) and hESCs (d). Cells were imaged at the indicated times (h:min) from telophase by phase-contrast microscopy (c) and from metaphase by differential interference contrast microscopy (d). Middle panel of (c) and upper left panel of (d): CETN1–GFP at centrosomes; enlargements and integrated intensity profiles show that the daughter cell with the older centrosome (c, upper; d, lower) inherits the MBd (lower right images in c,d). Mitotic midbody and MBds (c,d; arrows). MKLP1, MBd marker (red); α-tubulin, mitotic midbody and cell-boundary marker (green); DAPI, DNA (blue). Scale bars, 10 μm (c,d).