EMBO/soda-nel huggingface dataset:
PABPN1L Mediates Cytoplasmic mRNA Decay as a Placeholder during the Maternal-to-Zygotic Transition (2020)
Figure 4

Figure 4. PABPN1L is an RNA binding adapter of BTG4 involved in deadenylating maternal mRNAs.

A RNA immunoprecipitation (RIP) results showing the interaction of BTG4 with indicated transcripts, in the presence or absence of PABPN1L (full length, RRM-deleted, or R171A mutant). HeLa cells were co-transfected with plasmids expressing FLAG-tagged PABPN1L and HA-tagged BTG4 for 48 h before immunoprecipitating with an anti-HA antibody. RNAs recovered from the immunoprecipitants were subjected to RT-qPCR of the indicated transcripts. Fold change values of both input and IP samples were normalized by RIP results of HA-BTG4 and FLAG-PABPN1L co-expression groups. n = 3 biological replicates. Error bars, S.E.M. The P-value represents the two-tailed Student's t-test comparing the RIP results of BTG4 with the indicated transcripts in the presence of PABPN1L, *P < 0.05, **P < 0.01, and ***P < 0.001.

B Diagrams of mouse BTG4 constructs.

C, D Co-IP and western blot results showing interactions between PABPN1L and BTG4. Lysates from HeLa cells expressing HA-BTG4 (WT or mutants shown in (B)) and FLAG-PABPN1L were immunoprecipitated with an anti-HA antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.

E Diagrams of mouse BTG2 and BTG4 constructs, and co-IP results showing that PABPN1L binds to BTG4 but not BTG2.

F Diagrams and co-IP results showing BTG4 binding to PABPN1L. Lysates from HeLa cells expressing HA-BTG4 and FLAG-PABPN1L (full length and C-terminal deleted) were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.

G Diagrams of PABPN1 and PABPN1L constructs, and co-IP results showing that PABPN1L, but not PABPN1, binds to BTG4.

Data information: For all western blot results, at least three independent experiments were done with consistent results.
PABPN1L Mediates Cytoplasmic mRNA Decay as a Placeholder during the Maternal-to-Zygotic Transition (2020)
Figure 5

Figure 5. PABPN1L mediates BTG4 functions in vivo.

A Diagrams of the different treatments of (B).

B RT-qPCR results showing the relative expression level changes (MII/GV) of indicated transcripts. Fully grown GV oocytes of Pabpn1l-/- mice were microinjected with mRNAs encoding PABPN1L or BTG4 and were released from meiotic arrest at 12 h after microinjection. Total RNA was extracted from 10 oocytes in each sample. Error bars, S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test compared to the first column. ns: non-significant.

C Western blot results showing the expression of exogenous PABPN1L (full length, RRM-deleted, or R171A mutants) and BTG4 in Pabpn1l-/- oocytes at the MII stage. The total protein from 100 oocytes is loaded in each lane. pERK1/2 is blotted to indicate the developmental stages. DDB1 is blotted as a loading control. At least three independent experiments were done with consistent results.
PABPN1L Mediates Cytoplasmic mRNA Decay as a Placeholder during the Maternal-to-Zygotic Transition (2020)
Figure 6

Figure 6. Characterization of the PABPN1L poly(A) binding ability.

A-C Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly(A) variants (n = 6). The RNA substrate comprised 20 non-poly(A) nucleotides followed by a 10/20/30/60-adenosine poly(A) tail. PABPN1L-bound substrates were prepared with 2/1/0.5/0.2/0.1 RNA molecules per PABPN1L. The midpoint of the unfolding curve was determined as the melting temperature (Tm).

D RNA immunoprecipitation results showing the interaction of PABPN1L with indicated transcripts. Fully grown GV oocytes of WT mice were microinjected with mRNAs encoding PABPN1L (WT, RRM-deleted, or R171A mutant), and were released from meiotic arrest at 12 h after microinjection. Total RNAs were extracted from 350 MI stage oocytes in each sample. RNAs recovered from the immunoprecipitants were subjected to RT-qPCR of the indicated transcripts. Fold change values of both Input and IP samples were normalized by RIP results of the FLAG-PABPN1L microinjection group. Error bars, S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test comparing with RNA levels pulled-down by PABPN1L WT in the second column.
PABPN1L Mediates Cytoplasmic mRNA Decay as a Placeholder during the Maternal-to-Zygotic Transition (2020)
Figure 7

Figure 7. The role of PABPN1L in stabilizing BTG4 during oocyte maturation.

A Western blot results showing the levels of the indicated proteins in WT and Pabpn1l-/- oocytes. Total protein from 100 oocytes is loaded in each lane. DDB1 is blotted as a loading control.

B HeLa cells were transfected with plasmids expressing HA-BTG4 and FLAG-PABPN1L for 12 h and then treated by cycloheximide (CHX, 10 μM). Cells were harvested at the indicated time points for western blots. DDB1 is a loading control.

C Co-IP and western blot results showing the interaction of βTrCP1 with BTG4 in the presence or absence of PABPN1L.

D Co-IP and western blot results showing BTG4 polyubiquitination. HeLa cells transfected with plasmids encoding the indicated proteins were lysed and subjected to IP with an anti-HA affinity gel. Input cell lysates and precipitates are immunoblotted with the indicated antibodies.

E Western blot results showing endogenous BTG4 levels in WT and Pabpn1l-/- oocytes at the MII stage. FLAG-PABPN1L was expressed in Pabpn1l-/- oocytes by mRNA microinjected at the GV stage. Total proteins from 100 oocytes are loaded in each lane. ERK1/2 is blotted as a loading control.

F A schematic model for PABPN1L Function during the MZT. During oocyte maturation and fertilization, PABPN1L recruits BTG4 and CCR4-NOT deadenylase to the poly(A) tails of maternal transcripts and mediates cytoplasmic mRNA decay. At the molecular level, PABPN1L-Cter and BTG4-Cter2 mediate the BTG4-PABPN1L interaction, and arginine-171 determines the poly(A)-binding ability of PABPN1L. Furthermore, PABPN1L protects BTG4 from being polyubiquitinated by SCFβTrCP1 and degraded to create an expression window of BTG4 to mediate maternal mRNA decay. These events are prerequisites for ZGA. The gradient ribbons represent the indicated protein level and activity. Deep color represents a high level and activity. The functional domains of PABPN1L and BTG4 proteins are illustrated.

Data information: For all western blot results, at least three independent experiments were done with consistent results.

Expanded View Figure Legends
Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation (2014)
figf2

(a) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA (Scr) or Lamp2a mRNA–specific shRNA (shL2A) (left), and immunoblot analysis of LAMP-2A in sorted GFP+ T cells (right). Numbers above bracketed lines (left) indicate percent GFP+ cells. (b) Enzyme-linked immunosorbent assay of IL-2 and interferon-γ (IFN-γ) in TH1 cells transduced as in a and then left unstimulated (Rest) or stimulated for 24 h with anti-CD3 and anti-CD28 (Act). *P = 0.038 and **P = 0.031 (t-test). (c) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b, assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm (A495). *P = 0.013 (t-test). (d) Quantification of annexin V–positive (AnnV+) cells among resting or stimulated TH1 cells transduced as in a. Data are representative of three experiments (a) or are from four (IL-2) or five (IFN-γ) independent experiments (b) or four (c) or three (d) independent experiments (mean and s.e.m. in b–d).
Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation (2014)
figf3

(a) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti-CD3 and anti-CD28; LAMP-1 serves as a control. (b) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with (+) or without (−) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin (Lys inh); LAMP-1 serves as a control. (c) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence (NAc) or absence (Veh) of N-acetylcysteine. (d) Immunoblot analysis of lysates of TH1 cells transfected with GFP-Itch or GFP–mu-Itch (top) or with Myc–RCAN1 or Myc–mu-RCAN1 (bottom) and cotransfected with a GFP expression plasmid (to assess transfection efficiency), then activated for 24 h as in a. (e,f) Proliferation (e) and IL-2 production (f) of TH1 cells transfected with plasmid expressing GFP (Ctrl) or plasmids as in c and left unstimulated (Rest) or activated for 48 h (e) or 24 h (f) as in a (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.019 and **P = 0.0034 (e) or *P = 0.046 and **P = 0.044 (f) (t test). Data are representative of four (a,b), two (c) or three (d) independent experiments or are from three independent experiments (e,f; mean and s.e.m.).
Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation (2014)
figf4

(a) Immunoblot analysis of LAMP-2A in CD4+ T cells (left) and liver, kidneys, thymus and lungs (right) of L2A-cKO mice (KO) and their control littermates (Ctrl). (b) Quantitative PCR analysis of Lamp2a, Lamp2b and Lamp2c mRNA in T cells from L2A-cKO mice (right); results are presented relative to those of their control littermates (left), set as 1. (c) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form (LC3-I) and its autophagosome membrane–associated form (LC3-II) in total lysates of CD4+ T cells obtained from L2A-cKO mice or their control littermates and then left unstimulated (Rest) or activated for 24 h with anti-CD3 and anti-CD218 (Act), cultured for the final 4 h in the presence (+) or absence (−) of NH4Cl and leupeptin (NH4Cl+Leup). (d) Spleens from an L2A-cKO mouse and its control littermate (left), and flow cytometry of peripheral T cells from such mice (right). Numbers in quadrants (right) indicate percent CD4−CD8+ T cells (top left) or CD4+CD8− T cells (bottom right). (e,f) Proliferation (e) and IL-2 production (f) by CD4+ T cells isolated from the spleen of L2A-cKO mice and their control littermates and stimulated ex vivo for 48 h with anti-CD3 and anti-CD28 (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.0062 (e) or 0.006 (f) (t-test). (g) Immunoblot analysis of LAMP-2A, Itch and RCAN1 in total lysates of CD4+ T cells obtained from L2A-cKO mice (n = 3 (KO1, KO2 and KO3)) and their control littermates (n = 3) and activated for 24 h as in e,f. *, nonspecific band. (h) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4+ T cells isolated from L2A-cKO mice and their control littermates and activated for 24 h as in e,f; results are presented relative to those of resting cells. (i,j) Recall responses to OVA(323–339) in draining lymph nodes isolated from L2A-cKO mice and their control littermates after immunization with OVA(323–339), assessed as proliferation (i) and IL-2 production (j) (as in e,f). *P = 0.0054 (i) or 0.0052 (j) (t-test). (k,l) Recall responses to L. monocytogenes in CD4+ T cells isolated from L2A-cKO mice and their control littermates (n = 6 per group) previously uninfected (UI) or previously infected with L. monocytogenes (Inf), assessed as IFN-γ production (k) and proliferation (l) (as in e,f). *P = 0.032 (k) or 0.02 (l) (t-test). (m) L. monocytogenes in spleen and liver of L2A-cKO mice and their control littermates (n = 3 per genotype) immunized with L. monocytogenes and then reinfected with a high dose of L. monocytogenes. CFU, colony-forming units. *P = 0.008 and **P = 0.001 (t-test). Data are representative of two (a,g) or three (c) experiments or two experiments with six mice per genotype (d) or are from three (b,h), four (e,i,j), six (f) or two (k,l) independent experiments (mean and s.e.m. in b,e,f,h–l) or two experiments (m; mean and s.e.m. of triplicates).
Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation (2014)
figf5

(a) Immunoblot analysis of LAMP-2A in CD4+ T cells obtained from 4-month-old or 22-month old mice, then left resting or stimulated for 24 h with anti-CD3 plus anti-CD28. (b) Immunoblot analysis of RCAN1 and Itch in CD4+ T cells obtained from 4-month-old or 22-month old mice (n = 3 per group), then stimulated for 24 h as in a (left), and quantification of protein in 22-month old mice, presented relative to that of 4-month-old mice, set as 100%. *P = 0.044 (t-test). (c) Immunoblot analysis of LAMP-2A in total lysates of naive and memory CD4+ T cells left resting or activated for 24 h as in a. (d) Immunoblot analysis (bottom) of LAMP-2A in total lysates of naive and memory CD4+ T cells obtained from 4- or 22-month-old mice and left resting (R) or activated for 24 h as in a (A); above, quantification from results below (n = 3 mice per group). *P = 0.009 (t-test). (e) Electrophoretic mobility-shift assay of the binding of NFAT (N) and free probe (FP) in nuclear extracts of CD4+ T cells isolated from 3-, 12- or 22-month-old mice and activated for 4 h as in a. (f) ROS production in CD4+ T cells isolated from 4- or 22-month-old mice (n = 3 per group) and left resting or activated for 12 h as in a, assessed by flow cytometry and presented as the mean fluorescence intensity (MFI) of a fluorogenic probe for oxidative stress (CellROX Green). (g–i) Immunoblot analysis of LAMP-2A (g) and analysis of the proliferation (h) and IL-2 production (i) of TH1 cells obtained from 4- or 22-month-old mice and transduced with retrovirus expressing GFP alone (RV-GFP) or human LAMP-2A and GFP (RV-LAMP2A), sorted by GFP expression, cultured for 2 weeks and then stimulated for 24–48 h as in a (assessed as in Fig. 2c (h) or Fig. 2b (i)). *P 0.05 (analysis of variance with Tukey's post-test). (j) Immunoblot analysis of LAMP-2A in total lysates of CD4+ T cells obtained from two human donors (A and B) and left resting or activated for 24 or 48 h as in a (above lanes). (k) Real-time PCR analysis of LAMP2A mRNA in naive and memory CD4+ T cells obtained from young human donors (26 ± 2 years of age; n = 3) or old human donors (72 ± 6 years of age; n = 6) and activated for 24 h as in a; results were calculated by the change-in-cycle-threshold method (2−ΔCt) relative to those of the gene encoding β-actin. *P = 0.047 and **P = 0.0033 (Mann-Whitney). Data are representative of three independent experiments (a), three experiments (b,d,g) or two experiments (c,e,j) or are from three experiments (f,k) or four (h) or five (i) independent experiments (mean and s.e.m. in b,d,f,h,i,k).
The CRHR1/CREB/REST signaling cascade regulates mammalian embryonic neural stem cell properties (2022)
Figure 1

Figure 1. CRH signaling enhances embryonic neural stem cell properties.

A Neurosphere assays were performed using mouse E14.5 primary neural progenitors treated with CRH with or without antalarmin (Ant) (n = 3 biological replicates).

B Effects of CRH treatment on the stemness of neural progenitor cells at 2 days post-treatment were analyzed by Western blotting using an anti-SOX2 antibody (n = 3 biological replicates).

C, D Effects of CRHR1 overexpression on the (C) neurosphere formation and (D) SOX2 expression (n = 3 biological replicates).

E CRHR1 knockdown efficiency of shRNAs used in this study (n = 4 biological replicates).

F-I Effects of CRHR1 knockdown on the (F) neurosphere formation, (G) SOX2 expression, (H) neurosphere morphology, and (I) βIII-tubulin expression. n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).

J, K Immunostaining using anti-GFP (green) together with (J) anti-βIII-tubulin (clone TuJ1; red) (n = 5 biological replicates) or (K) anti-GFAP (red) antibodies (n = 3 biological replicates) after in vitro differentiation of E14.5 neural progenitor cells transduced with a lentiviral vector expressing shCRHR1.

Data information: Scale bars, 25 μm for (H), 50 μm for (A, C, F), and 100 μm for (J, K). Error bars represent SEM. Statistical significance was determined by unpaired Student's t-test (B, C, D, J, K), ordinary one-way ANOVA (A, E, F, G, H, I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.
The CRHR1/CREB/REST signaling cascade regulates mammalian embryonic neural stem cell properties (2022)
Figure 2

Figure 2. CRHR1 expression enhances neural stem cell properties in the developing brain.

A Embryonic brains that were intraventricularly electroporated CRHR1-expressing plasmids at E13.5 were harvested at E15.5 and immunostained using an anti-GFP (green) and SOX2 (red) antibodies.

B Quantification of the cell fraction in each laminar layer in (A) (n = 3 biological replicates).

C Percentage of SOX2+ cells among GFP+ cells in (A) (n = 4 biological replicates).

D Embryonic brains were electroporated with shCRHR1 plasmids with or without shCRHR1-resistant CRHR1 expression vectors at E14.5, and samples were harvested at E17.5 for anti-GFP immunolabeling. GFP+ cell localization was quantified (bottom panel) (n = 5 biological replicates).

E, F Brains electroporated with CRHR1 expression vectors at E13.5 were harvested at E15.5 and co-immunostained using anti-GFP (green) together with (E) anti-TBR2 (red) or (F) anti-CTIP2 (red) antibodies. n = 3 biological replicates.

G E14.5 Embryonic brains that were electroporated with CRHR1-expression vectors at E13.5 were pulse labeled with EdU and harvested at E15.5. Samples were then visualized for GFP (green), EdU (blue) and Ki67 (red) (n = 4 biological replicates).

Data information: Error bars represent SEM. Two-way ANOVA with Sidak's multiple comparisons test (B), unpaired Student's t-test (C, E, F, G) and two-way ANOVA with Tukey's multiple comparisons test (D) were used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. Scale bars, 20 μm. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. The upper and lower dashed lines in (A, D, E, F, G) indicate the boundaries between the CP and IZ, and IZ and VZ/SVZ, respectively.
The CRHR1/CREB/REST signaling cascade regulates mammalian embryonic neural stem cell properties (2022)
Figure 3

Figure 3. REST is a key transcriptional mediator of CRHR1 action in the embryonic neural stem cells

A, B Analysis of the effects of CRHR1 knockdown on neural stemness- and cell-fate-regulating transcription factors in neural progenitor cells by (A) qPCR (n = 4 biological replicates) and (B) Western blotting (n = 3 biological replicates) at 2 days post-transduction.

C, D The effects of CRHR1 overexpression on REST expression in neural progenitor cells analyzed by (A) qPCR (n = 4 biological replicates) and (B) Western blotting (n = 6 biological replicates).

E Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid with or without shREST vectors at E13.5 using anti-GFP (green) and SOX2 (red) antibodies.

F, G Quantification of (F) GFP+ cell distribution (n ≥ 4 biological replicates) and (G) percentage of SOX2+ cells (n ≥ 3 biological replicates) in (E).

H Embryonic brains were electroporated with shCRHR1 plasmids with or without REST expression vectors at E14.5, and brain samples were harvested at E17.5 for anti-GFP immunolabeling.

I Quantification of GFP+ cell localization in (H) (n ≥ 4 biological replicates).

Data information: Error bars represent SEM. Two-way ANOVA with Sidak's multiple comparisons test (A), unpaired Student's t-test (B, C, D), two-way ANOVA with Tukey's multiple comparisons test (F, I) and ordinary one-way ANOVA (G) were used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant. Scale bars, 20 µm. The upper and lower dashed lines in (E, H) indicate the boundaries between the CP and IZ, and IZ and VZ/SVZ, respectively.
The CRHR1/CREB/REST signaling cascade regulates mammalian embryonic neural stem cell properties (2022)
Figure 4

Figure 4. CREB activity is upregulated by CRHR1 expression

A, B Western blot analysis of E14.5 neural progenitor cells transduced with a lentiviral vector expressing (A) CRHR1 or (B) shCRHR1 using an anti-CREB and anti-phospho-CREB (pCREB) (S133) antibodies. n = 5 biological replicates.

C E14.5 neural progenitor cells were transduced with CRHR1-containing lentiviral vectors. At 2 days post-transduction, cells were treated with 10 µM H89 (PKA inhibitor) or 1 µM KN93 (CaM kinase inhibitor) for 8 h and harvested for Western blot analysis for CREB (top panel). Band intensities were quantified (bottom panel) (n = 3 biological replicates).

D Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid at E13.5 using anti-GFP (green) and pCREB (S133) (red) as primary antibodies. Immunostaining was quantified (right panel) (n = 5 biological replicates). White inset boxes were magnified and shown in the panels D' and D''.

E Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid with or without a vector expressing CREB S133A, a dominant negative mutant form of CREB, at E13.5 using anti-GFP (green) and SOX2 (red) antibodies.

F, G Quantification of (F) GFP+ cell distribution (n = 4 biological replicates) and (G) percentage of SOX2+ cells (n ≥ 3 biological replicates) in (E).

H Embryonic brains were electroporated with shCRHR1 plasmids with or without CREB expression vectors at E14.5, and then brain samples were harvested at E17.5 for anti-GFP immunolabeling.

I Quantification of GFP+ cell localization in (L) (n ≥ 4 biological replicates).

Data information: Error bars represent SEM. Unpaired Student's t-test (A, B, D), two-way ANOVA with Tukey's multiple comparisons test (F, I), and ordinary one-way ANOVA (C, G) were used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant. Scale bars, 20 µm for (D, E, H), 10 µm for (D' and D''). The upper and lower dashed lines in (D, E, H) indicate the boundaries between the CP and IZ, and IZ and VZ/SVZ, respectively.