EMBO/soda-nel huggingface dataset:
Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion (2020)
Figure 4

Fig 4. Pre-implantation progesterone (P4) supplementation is sufficient to normalize epithelial CCA and embryo invasion in Rb1d/d mice.

A, Serum estradiol-17β (E2) levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice (mean ± SEM, Student's t test; n=4 mice for each group).

B, Serum P4 levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice. In Rb1d/d mice, pre-implantation P4 treatment on days 2 and 3 increased serum P4 levels on day 4 but did not affect those on day 6 (mean ± SEM, Student's t test; n=4 mice for each group).

C, Protocol of pre-implantation P4 supplementation for the recovery of epithelial CCA in Rb1d/d mice. Rb1d/d mice underwent subcutaneous injection of P4 (2mg/day/mouse) on days 2 and 3, and sacrificed on day 4.

D, Persistent Ki67 expression in the epithelium of Rb1d/d mice on day 4 was recovered by pre-implantation P4 treatment on days 2 and 3. Scale bar = 100μm; le, luminal epithelium; s, stroma.

E, Ratio of Ki67-positive epithelial cells in Rb1d/d mice was completely suppressed by pre-implantation P4 supplementation on days 2 and 3. The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2D were used (mean ± SEM, Student's t test; n=5 different mice for each group). Three different high-powered fields per mouse were analyzed and each of the Ki67-posivie ratios was demonstrated.

F, Protocol of P4 treatment for the recovery of embryo invasion and subfertility in Rb1d/d mice. P4 (2mg/day/mouse) was injected to Rb1d/d mice on days 2-7 in Group 1, on days 2 and 3 in Group 2, and on days 5-7 in Group 3.

G, Resorption rate was reduced on day 8 in Groups 1 (n=41 different implantation sites) and 2 (n=30 different implantation sites), but not in Group 3 (n=40 different implantation sites). The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2I were used. H&E staining was performed using all sections that shows the implantation sites, and resorption rate was evaluated. *P<0.05 vs Rb1f/f mice; **P<0.05 vs Rb1d/d mice; Fisher's exact test.

H, Number of pups delivered by Rb1d/d dams was normalized by pre-implantation P4 treatment on days 2 and 3 (Group 2)(mean ± SEM, Student's t test). The same data of Rb1f/f and Rb1d/d dams without P4 treatment in Fig 1E were used. The numbers of pups in 20 Rb1f/f and 37 Rb1d/d dams without P4 treatment and 7 Rb1d/d dams with P4 treatment were evaluated.
Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion (2020)
Figure 5

Fig 5. Necroptosis of luminal epithelium at the implantation sites is observed in Rb1d/d mice.

A, pRIP3, a central mediator of necroptosis, was not expressed at the implantation sites of Rb1d/d mice but was expressed at those of Rb1f/f mice at 1500h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo; green dotted line, luminal epithelium; red dotted line, stroma.

B, TNFα stimulated the expression of pMLKL, a critical mediator of necroptosis, in the primary mouse uterine epithelial cells. Scale bar = 50μm; blue signal, nuclei stained by DAPI; red signal, pMLKL.

C, Uterine expression of Tnfα was elevated in the control mice (Rb1f/f mice) on days 5 and 6 compared to on day 4 (mean ± SEM, Student's t test; n=3 mice for each group). As for the uterine samples on days 5 and 6, those with implantation sites were examined.

D, The implanting embryo and the uterus produced TNFα at the implantation site of both Rb1f/f and Rb1d/d mice. Immunostaining of TNFα at the implantation sites of Rb1f/f and Rb1d/d mice were performed. Scale bar = 100μm; arrowhead, embryo.
Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion (2020)
Figure 6

Fig 6. CCA of uterine epithelial cells contributes to susceptibility to TNFα and TNFα-primed necroptosis through TNF receptor type 2.

A-B, In vitro analyses using primary mouse uterine epithelial cells demonstrated that TNFα induces the expression of annexin V, a marker of PS presentation at the outer membrane of Rb1f/f epithelial cells but does not in Rb1d/d ones, and P4 restores TNFα-primed annexin V expression in Rb1d/d uterine epithelial cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.

C-D, Annexin V assay using primary mouse uterine epithelial cells showed that TNFα administration increases the expression of annexin V in growth-arrested mouse epithelial cells with thymidine treatment, but does not in the control cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.

E, The expression of TNF receptor type 2 (TNFR2) was upregulated in growth-arrested epithelial cells with thymidine and P4 supplementation (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d groups, qPCR was performed in duplicate.
Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion (2020)
Figure 7

Fig 7. Pre-implantation P4 administration recovers epithelial necroptosis and trophoblast phagocytosis at the implantation site in Rb1d/d mice.

A, In Rb1d/d mice with pre-implantation P4 treatment, the fragmented uterine epithelial cells with the cytoplasmic lipid droplets were engulfed by the trophoblast cells. Scale bar = 2µm. Arrowhead, cytoplasmic fragments engulfed by trophoblast; dotted line circle, lipid droplets in the cytoplasm; tr, trophoblast; le, luminal epithelium; s, stroma; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.

B, Pre-implantation P4 treatment rescued the expression of MFG-E8 on day 5 of pregnancy in Rb1d/d uteri. Scale bar = 100μm. Blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; le, luminal epithelium; s, stroma; arrowhead, embryo.

C, Pre-implantation P4 treatment recovered the expression of pRIP3, a mediator of necroptosis, on day 5 of pregnancy in Rb1d/d uteri. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo.
Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion (2020)
Figure 8

Fig 8. Stromal Rb1 controls epithelial proliferation and embryo invasion in cooperation with epithelial Rb1.

A, RB was efficiently deleted in the uterine epithelium of uterine epithelium-specific conditional knockout mice (Rb1 eKO mice). Scale bar = 100μm. Rb1 eKO mice, Rb1loxP/loxPLtfCre/+ mice; Rb1 eControl mice (littermate controls of Rb1 eKO mice), Rb1loxP/loxP mice; le, luminal epithelium; s, stroma.

B, Number of new-born pups was comparable between Rb1 eKO and eControl dams (mean ± SEM, Student's t test; n=10 different dams for each group).

C, The expression patterns of Ki67 on day 4 in uterine epithelium were comparable between Rb1 eKO and Rb1 eControl mice. Scale bar, 100μm; le, luminal epithelium; s, stroma.
Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion (2020)
Figure 9

Fig 9. CCA of uterine luminal epithelial cells is a hallmark of embryo implantation in mice and humans.

A, Representative pictures of Ki67 staining in the human endometria obtained from women at the time of embryo implantation. Samples were divided into two groups according to the outcome of clinical pregnancy following endometrial biopsy. Scale bar = 100μm; le, luminal epithelium; s, stroma.

B, Ratio of Ki67-positive cells in the human uterine luminal epithelium during the implantation period was lower in the group with pregnancy than the group without pregnancy (mean ± SEM, Student's t test; n=10 different individuals for each group). Three different high-powered fields per sample were analyzed and each of the Ki67-posivie ratios was demonstrated.

C, Potential processes that contribute to embryo invasion through uterine RB-induced CCA and necroptosis of luminal epithelial cells.

Expanded View Figure Legends
The p38-interacting protein p38IP suppresses TCR and LPS signaling by targeting TAK1 (2020)
Figure 1

Figure 1. p38IP inhibits TCR- and LPS-induced cytokine production

(A-C) Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) and REACTOME Pathways (27.02.2019) enrichment analyses of differentially expressed genes in stimulated sh-NC vs sh-p38IP Jurkat E6.1 T cells (fold change >2, P<0.05), showing the top 8 significantly enriched terms.

(D) Heatmap showing the expression change of representative NF-κB target genes from the genes with significantly changed expression (fold change >2). The data represent the log2 (gene FPKM) normalized for each row. RNA-seq data is from 2 independent experiments.

(E) Intracellular cytokine staining assay of human TNF-α and IL-2 in sh-NC and sh-p38IP Jurkat E6.1 cells stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for 6 hours (average frequency of positive staining YFP+ cells in sh-NC cells without stimulation = 1).

(F) ELISA analysis of IL-2 and IL-6 production in the culture supernatants of human PBMCs transfected with si-NC or si-p38IP, and then stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 (αCD3/CD28) for 24 hours.

(G and H) Real-time PCR analysis of TNF-α, IL-6 and IL-1β (G) and of IFN-β, ISG54 and ISG56 (H) mRNA production in RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times (average mRNA production in si-NC cells at 0 hour = 1).

(I) ELISA analysis of TNF-α, IL-6, IL-1β and IFN-β production in the culture supernatants of human PBMCs transfected with si-NC and si-p38IP and then stimulated with 1μg/mL LPS for 24 hours.

Data information: **P < 0.01, ***P < 0.001, ****P < 0.0001; n.d., not detectable; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.). Data are representatives of two (F, I) and three (E, G-H) independent experiments.
The p38-interacting protein p38IP suppresses TCR and LPS signaling by targeting TAK1 (2020)
Figure 2

Figure 2. Knockdown of p38IP enhances TCR- and LPS- induced activation of p38 and NF-κB

(A) Immunoblot analysis with the indicated antibodies of total cell lysates of sh-NC and sh-p38IP Jurkat E6.1 cells stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times. Actin served as a loading control (throughout).

(B) Statistical analysis of p65 nuclear translocation of sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated with 50 ng/ml PMA plus 1µM ionomycin for the indicated times. p65 nuclear translocation was indicated by the fluorescence intensities ratio between the nuclear p65 and the whole cell p65 from each cell. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).

(C) Luciferase reporter assay of sh-NC and sh-p38IP Jurkat E6.1 cells transfected with NF-κB luc plus an internal control Renilla luciferase reporter for 36 hours, followed by stimulation with ( + ) or without ( - ) 50ng/ml PMA plus 1 µM ionomycin for 8 hours. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).

(D,E) Immunoblot analysis with the indicated antibodies of total cell lysates of RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times.

Data information: Data in (A-E) are representatives of at least three independent experiments.
The p38-interacting protein p38IP suppresses TCR and LPS signaling by targeting TAK1 (2020)
Figure 4

Figure 4. p38IP dynamically interacts with TAK1 following stimulation

(A) Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for the indicated times, the cell lysates were immunoprecipitated and immunoblotted as indicated. The relative intensity of the IP bands was quantified by densitometry and is shown below the graph.

(B) HEK293T cells transfected with Flag-tagged TAB2 (open arrow) or TAB2-ub (solid arrow) were lysed and immunoprecipitated with anti-Flag, and then immunoblotted with the indicated antibodies. The asterisk indicates a non-specific band. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.

(C) HEK293T cells co-transfected with HA-tagged p38IP together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow) were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. Asterisk indicates non-specific bands. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.

(D) HEK293T cells co-transfected with HA-tagged p38IP, Flag-tagged TAB2-ub, together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow), were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.

Data information: Data are representatives of three (B-D) and four (A) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).

(E) Schematic of dynamic association between p38IP and TAK1 after stimulation.
The p38-interacting protein p38IP suppresses TCR and LPS signaling by targeting TAK1 (2020)
Figure 5

Figure 5. In vitro analysis of K63-linked polyUb binding, transferring and influencing protein binding affinity

(A) GSH-Sepharose beads-immobilized full length or truncated GST-TAK1 proteins were incubated with K63-linked polyUb chains in 300 μl binding buffer, followed by washing and resolving in SDS-PAGE, and then immunoblotted with anti-ub. GST-fused NEMO serve as positive control and NEMO L329P mutant as negative control. The blotted membrane was stained with Ponceau S to demonstrate the amount of the input GST-fused proteins (lower panel).

(B) Strategy for in vitro polyUb transfer experiments.

(C and D) Immobilized GST-TAB2 was pre-incubated with K63-linked polyUb chains and then eluted from beads, followed by incubation with eluted GST-TAK1 and GST-p38IP as shown in (B). Incubation mixture were immunoprecipitated with anti-TAB2 or anti-TAK1, and then immunoblotted with anti-ub, anti-TAB2, anti-TAK1 and anti-p38IP.

(E) Statistical analysis of polyUb binding to TAB2 or TAK1 in (C and D). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).

(F) Immobilized GST-TAB2 and GST-TAK1 were respectively pre-incubated with K63-linked polyUb chains or not as indicated, then eluted from beads, followed by coincubation as indicated. Incubation mixture were immunoprecipitated with anti-TAB2, and then immunoblotted with anti-TAB2 and anti-TAK1. The relative intensity of immunoprecipitated TAK1 was calculated and shown in the right. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).

(G) Immobilized GST-TAK1 was pre-incubated with K63-linked polyUb chains or not, then eluted from beads, followed by incubation with eluted GST-p38IP. Incubation mixture were immunoprecipitated with anti-TAK1, and then immunoblotted with anti-TAK1 and anti-p38IP. The relative intensity of immunoprecipitated p38IP was calculated and shown in the right. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).

Data information: Data are representatives of at least three independent experiments.
The p38-interacting protein p38IP suppresses TCR and LPS signaling by targeting TAK1 (2020)
Figure 6

Figure 6. The T1B (215-275 aa region) of p38IP is responsible for its interaction with TAK1

(A) Schematic diagram of the truncations of p38IP, in which the numbers indicate the residues of the truncations and the red box indicates T1B.

(B) HEK293T cells transfected with N-terminal (left panels) or C-terminal (right panels) deletion mutants of p38IP as indicated in (A) were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1.

(C) Ribbon representation of a model structure of the p38IP-T1B (pink) and TAK1 (green).

(D) GST pull down assay. 3μg recombinant GST-T1B protein which immobilized on glutathione Sepharose beads was incubated with the cell lysates that obtained by adding 1ml lysis buffer to the HEK293T cells in 10 cm cell culture dish with a final protein concentration around 1μg/μl, after 4 hrs incubation, washed beads with cell lysis buffer five times and subjected to SDS-PAGE for immunoblotting with anti-TAK1.

(E) HEK293T cells transfected with YFP-p38IP T1B were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1.

(F) Jurkat TAg cells transfected with YFP vector or YFP-T1B were stimulated with 10μg/ml anti-CD3 for indicated times, then lysed and immunoprecipitated with anti-TAK1, followed by immunoblotting with the indicated antibodies.

(G) Primary human PBMCs transfected with YFP vector or YFP-T1Bwere stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 for the indicated times, then lysed and analyzed by immunoblotting.

(H) Intracellular cytokine staining analysis of TNF-α (upper) and IL-2 (lower) in primary human PBMCs transfected and stimulated as in (F) for 3 hours (TNF-α) and 8 hours (IL-2).

Data are representatives of three independent experiments. ***P < 0.001, (two-tailed unpaired t-test, mean and s.e.m.).
The p38-interacting protein p38IP suppresses TCR and LPS signaling by targeting TAK1 (2020)
Figure 7

Figure 7. p38IP scaffolds USP4 to deubiquitinate TAK1

(A) HEK293T cells co-transfected with YFP-p38IP and Flag-vector, Flag-USP3 or Flag-USP4 as indicated were lysed and immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.

(B) HEK293T cells transfected with Flag-USP4 and YFP-vector or YFP-p38IP as indicated were lysed and immunoprecipitated with anti-IgG or anti-TAK1 and then immunoblotted with anti-Flag and anti-TAK1.

(C) HEK293T cells transfected with plasmids as indicated were lysed and immunoprecipitated with anti-TAK1, followed by in vivo ubiquitination assay of TAK1 as described in methods.

(D) In vitro deubiquitination assay of TAK1 as described in methods.

(E) sh-NC and sh-p38IP Jurkat E6.1 cells transfected with si-NC, si-p38IP, Flag or Flag-USP4 as indicated were stimulated with anti-CD3/CD28 for 0 and 15 min at 48 hours post transfection, and then lysed and immunoprecipitated with anti-TAK1 under stringent conditions and immunoblotted with anti-ub. Statistical analysis of p-p38 levels was shown below the blot. **P < 0.01; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).

(F) Jurkat E6.1 T cells were stimulated with anti-CD3/CD28 for the indicated times. The cell lysates were immunoprecipitated with anti-p38IP or anti-TAK1 and then immunoblotted with the indicated antibodies.

(G) sh-NC and sh-p38IP Jurkat E6.1 cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times, and then lysed and immunoprecipitated with anti-TAK1 and immunoblotted with anti-USP4 and anti-TAK1.

(H) Mapping the USP4 binding domain of p38IP.

(I) Overall structure of p38IP U4B (gold) and USP4 (deep teal).

(J) Structural comparison between CYLD (blue) and USP4 (deep teal), the fingers subdomain was shown in red.

(K) Schematic illustration of the molecular mechanism underlying p38IP regulation of TAK1. In quiescence state, p38IP constitutively competes with TABs for binding to TAK1. Upon receptor stimulation, p38IP transiently dissociates from TAK1, and increases its association with USP4, which might due to the phosphorylation of p38IP (indicated by an asterisk in the figure). Once TAK1 is activated, p38IP not only reassociates with TAK1, disassembling TABs-TAK1 complex and blocking the transfer of unanchored K63-linked polyUb chains from TAB2 to TAK1, but also specifically recruits USP4 to remove the unanchored K63-linked polyUb binding or conjugated ubiquitination of TAK1.

Data information: Data (A-H) are representatives of three independent experiments.

Expanded View Figure legends
The Autophagy Receptor TAX1BP1 (T6BP) improves antigen presentation by MHC-II molecules. (2022)
Figure 2

Figure 2. T6BP silencing influences the stability of HLA αβ dimers, mildly their cell-surface expression levels but has no impact on their internalization kinetics.

(A) Cell-surface expression of MHC-II molecules was assessed using flow cytometry. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48h post-treatment, HLA-DR molecules were detected using L243 (left panels) and Tü36 antibodies (right panels) that recognize mature (HLA αβ heterodimers) and both mature and immature (HLA αβ and αβIi complexes), respectively. Left, MFI of one representative experiment is presented as histogram. Light grey lines: isotype negative controls; black lines and filled grey lines: anti-MHC-II staining of siCTRL- and siT6BP-treated cells, respectively. Right, five biological replicates were combined and presented as the means (+/- SD) of mean fluorescent intensity (MFI) standardized to the control conditions. CTRL: control. Mann-Whitney's test; * p<0.05; # p>0.05.

(B) and (C) internalization kinetics of cell-surface HLA-DR molecules. HeLa-CIITA cells were transfected as above. 48h post-treatment, mature HLA-DR molecules were stained at 4°C using L243 antibody. Cells were then incubated at 37°C (B) or at 4°C (C). At indicated time-points, cells were stained with a fluorescent secondary antibody at 4°C. Results are represented as MFI of MHC-II molecules stained with the L243 antibody remaining at the cell surface (B and C, left panels), or as percentage (%) of MFI relative to time 0h (100%) (B and C, right panels). Results are presented (+/- SD) of technical replicates and representative of three independent experiments.

(D) T6BP silencing affects the formation of stable MHC-peptide complexes. HeLa-CIITA cells were transfected as above and pulse-labeled with 35S-Met/Cys for 30 min, washed and chased for 4h at 37°C. MHC-II molecules were then immunoprecipitated using TÜ36 antibody and analyzed on SDS-PAGE after incubation of the immunoprecipitated protein complexes with SDS at 95°C (B: boiled) or room temperature (NB: non-boiled) to visualize α, β and Ii chains and SDS resistant αβ dimers, respectively. The bands corresponding to α, β and Ii chains are indicated. The arrow indicates the SDS-resistant stable HLA αβ heterodimers. This gel is representative of two independent experiments.