EMBO/soda-nel huggingface dataset:
The scaffold protein EPG-7 links cargo-receptor complexes with the autophagic assembly machinery. (2013)
Figure 7.

EPG-7 is not required for starvation-induced autophagic degradation of protein substrates. (A and B) C17E4.2::GFP is weakly expressed and diffusely localized in a wild-type embryo. (A) DIC image of the embryo shown in B. (C and D) C17E4.2::GFP is expressed at dramatically elevated levels and accumulates into many aggregates in lgg-1 (C) and epg-7 (D) mutant embryos. (E and F) W07G4.5::GFP is expressed in the intestine and a few aggregates are formed in a wild-type embryo at the fourfold stage. (E) DIC image of the embryo shown in F. (G and H) W07G4.5::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in atg-3 (G) and epg-7 (H) mutants. Images in B–D or F–H were taken using the same exposure time. (I) Levels of C33D9.6::GFP and W07G4.5::GFP are dramatically elevated in atg-3 and epg-7 mutant embryos. (J and K) C17E4.2 aggregates in atg-3 mutants overlap with EPG-7 aggregates (J), but are separable from SQST-1 aggregates (K). Insets show a magnified view. (L) Colocalization frequency of C17E4.2, C33D9.6, and W07G4.5 aggregates with EPG-7 and SQST-1 aggregates in atg-3 mutants. (M) SQST-1::GFP accumulates in hypodermal cells in epg-7 mutants at the larval stage. (N) SQST-1::GFP aggregates disappear in rheb-1(RNAi);epg-7 mutant larvae. (O) Dramatic accumulation of SQST-1::GFP aggregates in hypodermal cells in atg-3 mutant larvae. (P) SQST-1::GFP aggregates persist in rheb-1(RNAi);atg-3 mutant larvae. (Q–T) At larval stages, W07G4.5::GFP forms aggregates in intestinal cells (Q and R). These aggregates disappear upon RNAi knockdown of let-363 (S and T). (U–X) W07G4.5::GFP aggregates accumulate in intestinal cells in epg-7 mutant larvae (U and V). These aggregates disappear when let-363 is simultaneously depleted (W and X). (Y and Z) In atg-3 mutants, W07G4.5::GFP aggregates accumulate in intestinal cells (Y) and persist after RNAi knockdown of let-363 (Z). (A2) Levels of W07G4.5::GFP in wild type, epg-7 mutants, and atg-3 L1 larvae before and after 6 h starvation treatment. (B2) Model for the role of epg-7 in degradation of SQST-1 aggregates. SQST-1 directly interacts with EPG-7 and is recruited into EPG-7 aggregates. EPG-7 also associates with multiple Atg proteins, which may be recruited to the SQST-1–EPG-7 complex in a hierarchical order. IM, isolation membrane. (C2) Hierarchical relationship of the scaffold proteins EPG-2 and EPG-7 and other essential ATG and EPG proteins in the aggrephagy pathway. Bars: (A–H, J, and K) 10 µm; (J and K, insets) 5 µm; (M–Z) 10 µm.
A peptide encoded by pri-miRNA-31 represses autoimmunity by promoting Treg differentiation (2022)
Figure 1

Figure 1. miPEP31 is an endogenous peptide encoded by pri-miR-31

(A) Schematic representation of miPEP31 translation. Pri-miR-31 transcribed from the miR-31 host gene. sORF was found by the ORF Finder. The yellow box indicates the ATG of the sORF. The red line indicates the sequence of miR-31. And the green line indicates the location of sORF on pri-miR-31.

(B) The nucleotide and amino acid sequences of the sORF.

(C) Ribo-seq data of NIH 3T3 cells (SRR6023964) were analyzed and visualized by IGV. Ribo-seq reads were found on pri-miR-31. The red box indicates the reads on partial of the coding region of miPEP31.

(D) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp (without ATG) to the sequence from 5' cap of pri-miR-31 to ATG site or which mutated to ATT of the sORF. Cells were selected by G418 for 2 days. Analysis of the GFP expression by confocal microscopy or flow cytometry, scale bar 100 μm.

(E) Immunoblot analysis of GFP and miPEP31 in NIH 3T3 cells transfected by pEGFP-N1-miPEP31 or pEGFP-N1, the red arrow indicate predicted size of miPEP31-GFP band.

(F) Flow-fish to detect the localization of pri-miR-31. The location of pri-miR-31 was analyzed by confocal microscopy. Scale bar 5 μm.

(G) Immunoblot analysis of multiple mouse tissues with a specific antibody against miPEP31. Values are expressed as fold changes relative to controls and normalized to GAPDH, the red arrow indicate predicted size of miPEP31.

(H) Immunoblot analysis of spleen, lymph node and thymus from WT or miR-31-/- mice with miPEP31 antibody, the red arrow indicate predicted size of miPEP31.
A peptide encoded by pri-miRNA-31 represses autoimmunity by promoting Treg differentiation (2022)
Figure 3

Figure 3. miPEP31 is cell-penetrating peptide both in vitro and in vivo.

(A) CD4+ T cells were activated with anti-CD3/28 and treated with different concentration of FAM-miPEP31 for 24 hours, the fluorescein was detected by Flow Cytometry.

(B) Confocal Microscopy was used to analyze the fluorescence of FAM-miPEP31 in anti-CD3/28 activated CD4+ T cells treated with 10 μM FAM-miPEP31. Scale bar 5 μm.

(C-D) Anti-CD3/28 activated CD4+ T cells were cultured with 10 μM miPEP31 for 24 hours. (C) Western Blotting analysis of Cytochrome C and Histone H2A in total lysates and nuclear lysates. (D) ELISA to detect the concentration of miPEP31 in protein of total cell and nuclear extracts in CD4+ T and Treg cells treated with or without 10 μM miPEP31. Data in (D) are presented as mean ± SEM of four biological replicates. ns, not significant (CD4+ T versus Treg), unpaired two-tailed Student's t-test..

(E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, FAM-labeled miPEP31 was added. Imaging Flow Cytometry analysis of FAM labeled miPEP31 in CD4+ T cells. Scale bar 7 μm.

(F, G) C57BL/6 mice were injected intravenously with 100 μg FAM-labeled miPEP31. Splenocytes were isolated and stained for CD4 and DAPI. Imaging Flow Cytometry analysis of miPEP31 in CD4+ T cells (F) and non-CD4 splenocytes (G) , scale bar 10 μm.
Dual Regulatory Actions of LncBMP4 on BMP4 promote Chicken Primordial Germ Cell Formation (2021)
Figure 2

Figure 2 LncBMP4 promotes the origination of PGCs by targeting BMP4, in vitro.

A. Up: The schematic diagram of PGCs induced from chicken ESCs in vitro. Down: Morphological observation of the number of embryoid bodies in the in vitro PGCs induction model after LncBMP4overexpression and interference; oeLncBMP4 indicates the overexpression of LncBMP4; shLncBMP4 indicates the interference of LncBMP4; BMP4 induction was regarded as the control. Scale bar: 60 µm (n = 3 independent experiments).

B. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR after LncBMP4 overexpression and interference, in vitro (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA) .

C. The efficiency of PGCs differentiation was detected by IF 4 d after induction, using C-KIT and CVH antibodies. Scale bar: 60 µm (n = 3 independent experiments).

D. Detection of PGCs formation efficiency after LncBMP4 overexpression or interference, in vitro, by flow cytometry using the CVH antibody (Data are shown as mean ± SEM, n = 3 independent experiments, **** p < 0.0001, one-way ANOVA. Gating strategies and Blank were supplied in Appendix Figure S14A-D).
Dual Regulatory Actions of LncBMP4 on BMP4 promote Chicken Primordial Germ Cell Formation (2021)
Figure 3

Figure 3 LncBMP4 regulates the migration of PGCs in vivo.

A. The results of IHC examining using anti-DDX4 antibody shows that the overexpression of LncBMP4 increases the number of PGCs in the genital ridge, and interference with LncBMP4 reduces the number of PGCs in the genital ridge. The number of PGCs was counted through the Segmentation module of Image J software. Scale bars:200 μm (top row),40 μm (bottom row) (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA).

B. The number of PGCs in the genital ridge, following the overexpression or interference of LncBMP4, as determined by flow cytometry (Data are shown as mean ± SEM, n = 3 independent experiments, **** p < 0.0001, one-way ANOVA).

C. The number of PGCs labeled with PkH26 (red) that migrated to the genital ridge in a frozen section, the number of PGCs labeled with PkH26 (red) was counted using Image J software. Scale bar: 70 µm (Data are shown as mean ± SEM, n = 6 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).

D. The effect of overexpression and interference of LncBMP4 on the proliferation of PGCs was detected by EDU proliferation assay. Scale bar: 100 µm (n = 3 independent experiments).
Dual Regulatory Actions of LncBMP4 on BMP4 promote Chicken Primordial Germ Cell Formation (2021)
Figure 4

Figure 4 gga-mir-12211 regulates the formation of PGCs by targeting LncBMP4 and BMP4.

A~B. Screening of miRNAs that target both BMP4-3'UTR and LncBMP4. (A) Two miRNAs (gga-mir-12211和gga-mir-12258) simultaneously target BMP4-3'UTR and LncBMP4. (B) Schematic diagram of base sequence and structure of gga-mir-12211 and gga-mir-12258.

C. Morphological observation of the number of embryoid bodies in the PGC in vitro induction model after the overexpression and interference of gga-mir-12211. BMP4 induction was regarded as control. Scale bar: 60 µm (n = 3 independent experiments).

D. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR in the genital ridge after gga-mir-12211 overexpression and interference in vitro (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA).

E. The efficiency of PGC differentiation was detected by IF, 4 d after induction, using C-KIT and CVH antibodies. Scale bar: 60 µm (n = 3 independent experiments).

F. Detection of PGCs formation efficiency after the overexpression or interference of gga-mir-12211, in vitro, as assessed by flow cytometry using the CVH antibody (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA.).
Dual Regulatory Actions of LncBMP4 on BMP4 promote Chicken Primordial Germ Cell Formation (2021)
Figure 5

Figure 5 m6A modification promotes LncBMP4 as a ceRNA, to regulate the expression of BMP4.

A. A luciferase reporting system was used to detect the regulatory effects of gga-mir-12211 on the activity of BMP4-3'UTR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA).

B. qRT-PCR was used to detect the regulatory effects of gga-mir-12211 on LncBMP4 expression (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001, **** p < 0.0001, one-way ANOVA).

C. The results of the luciferase reporting system showed that LncBMP4 could rescue the gga-mir-12211 inhibitory effects against BMP4-3'UTR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA).

D. Distribution diagram of RRACH sites on LncBMP4, BMP4-3'UTR, and gga-mir-12211.

E and F. Enrichment levels of m6A in LncBMP4 (E) and BMP4-3'UTR (F) were detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).

G. The effects of MA and NPC on m6A enrichment levels in the P2 region of LncBMP4 were detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001, **** p < 0.0001, one-way ANOVA.).

H and I. The effects of m6A modifications of LncBMP4 on the ceRNA system were detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA.).
Dual Regulatory Actions of LncBMP4 on BMP4 promote Chicken Primordial Germ Cell Formation (2021)
Figure 6

Figure 6 m6A regulates ceRNA, in a dose-dependent manner, at the RRACH sites of LncBMP4.

A. The influence of m6A on the stability of LncBMP4 was detected by qRT-PCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).

B. The effects of m6A modification on the binding ability of LncBMP4 with gga-mir-12211 was detected using a luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA).

C. Effect of NPC and MA treatment on the activity of Luc-LncBMP4 was detected using a luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).

D. The enrichment level of m6A, for LncBMP4 variants with the progressive accumulation of RRACH site mutations, was detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, one-way ANOVA).

E. The ability of LncBMP4, modified with different levels of m6A, to bind gga-mir-12211 was detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).
Dual Regulatory Actions of LncBMP4 on BMP4 promote Chicken Primordial Germ Cell Formation (2021)
Figure 7

Figure 7 EPC5, encoded by LncBMP4, exhibits similar functions as LncBMP4.

A. Morphological observation of small peptides and LncBMP4 backbone chains, promoting PGC formation in vitro, Scale bar: 60 µm (n =3 independent experiments).

B. qRT-PCR was used to detect the expression of related genes in each group, during induction (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA).

C. The efficiency of PGC differentiation was detected by IF after 4 d of induction using C-KIT and CVH antibodies. Scale bar: 60 µm (n =3 independent experiments).

D. The number of CVH-positive cells was determined by flow cytometry (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA.).
Dual Regulatory Actions of LncBMP4 on BMP4 promote Chicken Primordial Germ Cell Formation (2021)
Figure 8

Figure 8 m6A inhibits LncBMP4 translation into EPC5, to regulate BMP4 transcription.

A. The binding of the small peptide EPC5 with the BMP4 promoter region was detected by ChIP-qPCR in ESCs and PGCs (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).

B. Double-luciferase detection of small peptides that affect BMP4 transcription (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA).

C. Effects of m6A on small peptide expression, assessed by western blot (Up), Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).

D and E. Western blot(Up) was used to detect gga-mir-12211 and m6A affect the ability of LncBMP4 to translate EPC5. Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001,**** p < 0.0001, one-way ANOVA.).
Interplay of LRRK2 with chaperone-mediated autophagy (2013)
figf1

(a) Top: immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of changes in total LRRK2 (n = 6 independent experiments). SH-SY5Y cells were untreated or incubated with 5 μM lactacystin (Lact), 10 mM 3MA or 20 mM NH4Cl and 100 μM leupeptin (NL) for 9.5 h. (b) Top: representative immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of LRRK2 relative to that in untreated samples (n = 3 or 4 independent experiments). Brain slices from mouse cortex, midbrain or cerebellum were incubated without additions or with NL or 3MA for 2 h at 37 °C. (c,d) Homogenate (Hom) and lysosomal fractions (Lys) isolated from SH-SY5Y cells (c) or the indicated mouse brain regions (d), immunoblotted for the indicated proteins. (e) HEK293 cells expressing GFP-tagged LRRK2 and transduced with lentivirus control (Ctr) or carrying shRNA against LAMP-2A (L2A(−)), incubated or not with NL for 12 h and immunoblotted for the indicated proteins. (f) Immunoblots for LRRK2 and LAMP-2A of homogenates and lysosome-enriched fractions isolated from control fibroblasts (Ctr) or fibroblasts stably interfered for LAMP-2A (L2A(−)), maintained in the presence or absence of serum. Actin and LAMP-1 (L-1) are shown as loading controls of homogenate and lysosomes, respectively. All values are mean + s.e.m.; *P 0.05, unpaired t-test. Full-length blots and gels in Supplementary Figure 12.
Interplay of LRRK2 with chaperone-mediated autophagy (2013)
figf2

(a) Amino acid locations of the putative CMA targeting motifs in LRRK2. (b) Binding, association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers, untreated or pretreated with protease inhibitors (PI) and incubated with LRRK2. Inset: representative immunoblot. LAMP-2A is shown as lysosomal loading control. Input (Inp), one-fifth of LRRK2 added. n = 7 independent experiments. Uptake: difference between LRRK2 associated with lysosomes with and without PI. (c) Effect of increasing concentrations of LRRK2 on the degradation of a radiolabeled pool of cytosolic proteins by intact lysosomes. Values are percentage of the degradation without LRRK2 (n = 5 or 6 independent experiments). (d) Association of GAPDH to rat liver lysosomes in the presence of increasing concentrations of LRRK2. Values are percentage of the GAPDH associated with lysosomes in the absence of LRRK2 (n = 4–6 independent experiments in duplicate). Inset: representative immunoblot. Inp: 1 μg of GAPDH. (e,f) Association of LRRK2 and RNase A (e) or LRRK2 and GAPDH (f) with PI-treated rat liver lysosomes incubated with increasing concentrations of GAPDH (e) or RNase A (f) or ovalbumin (Ov). Left: representative immunoblots. Right: protein association expressed as a percentage of the value when incubated with lysosomes alone (n = 5 or 6 independent experiments). Inp: RNase A (0.4 μg), LRRK2 (0.04 μg) or GAPDH (1 μg). (g,h) Association of LRRK2 to lysosomes incubated with GAPDH (g) or RNase A (h) at 4 °C (n = 3 independent experiments). (i,j) Immunoblots of brain lysosomes incubated with LRRK2 and increasing concentrations of GAPDH (i) or RNase A (j). All values are mean + s.e.m.; differences with lysosomes incubated with the protein alone were significant at *P 0.05, unpaired t-test. Full-length blots and gels in Supplementary Figure 12.
Interplay of LRRK2 with chaperone-mediated autophagy (2013)
figf3

(a) Top: immunoblot for GFP of tet-on HEK293 cells expressing GFP-tagged WT or G/S LRRK2 pulsed 24 h with doxycycline and, 48 h later (no longer undergoing de novo synthesis), incubated without additions (−) or with 20 mM NH4Cl and 100 μM leupeptin (NL), 5 μM MG132 (MG) or 10 mM 3MA. Bottom: percentage inhibition (n = 3 independent experiments). (b) Same cells as in a, untreated (Ctr) or treated with RNA interference for LAMP-2A (L2A(−)), pulsed as in a, sequentially collected and immunoblotted. Top left panels: representative immunoblots for GFP. Actin is shown as a loading control. Bottom left panels, quantification, corrected by levels of knockdown and loading control (n = 3 or 4 independent experiments). Top right: immunoblot for LAMP-2A. Bottom right: percentage of LRRK2 degraded per hour in the same samples. (c) Binding, association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers, untreated or pretreated with protease inhibitors (PI) and incubated with WT or G/S LRRK2. Inset: representative immunoblot (n = 9 independent experiments). (d) Association of GAPDH (5 μg) to starved rat liver lysosomes in the presence of increasing concentrations of WT or G/S LRRK2. Inset: representative immunoblot n = 4–6. (e,f) Binding of WT and G/S LRRK2 to lysosomes in the presence of GAPDH (e) or RNase A (f). Top: representative immunoblots. Bottom: lysosome-bound LRRK2 expressed as a multiple of the amount bound when added alone. Trends of mean values (n = 2 independent experiments); error bars indicate range. (g) Immunoblot of rat brain lysosomes (Lysosom) incubated with WT or G/S LRRK2 as in c. Inputs, one-tenth of protein added. (h,i) Rat brain lysosomes incubated as in d and e, respectively. Left: representative immunoblots. Right: quantifications (n = 3 or 4 independent experiments). All values expressed as mean + s.e.m.; differences with untreated samples (*) and between WT LRRK2 and G/S LRRK2 (§) were significant at P 0.05; n.s., not significant; ANOVA and Bonferroni test. Full-length blots and gels in Supplementary Figure 12.
Interplay of LRRK2 with chaperone-mediated autophagy (2013)
figf4

(a) Proteolysis in HEK293 cells stably transfected with an empty plasmid (Ctr) or plasmids expressing WT or G/S LRRK2 under the control of a tet promoter, treated with doxycycline to activate protein expression and then labeled with [3H]leucine for 48 h. After extensive washing, protein degradation at the indicated times was measured, in cells maintained either in the presence (+) or absence (–) of serum, as the amount of acid-precipitable radioactivity (amino acids and small peptides) released into the media. Values are expressed as percentage of radioactivity in proteins (acid precipitable) at time 0 (n = 4 independent experiments, each in triplicate). (b) The same cells, transiently transfected with a KFERQ-PA-mCherry1 fluorescent reporter for CMA activity. After photoactivation, cells were maintained for 16 h in the presence or absence of serum. Left: representative micrographs; right column shows higher magnification images of outlined regions. Arrows denote puncta. Right: average number of puncta per cell in each condition, measured in >150 cells. (c) Cultured ventral midbrain dopaminergic neurons from Ctr and from WT-LRRK2 and G/S-LRRK2 transgenic mice, transduced with the same reporter as in b and co-stained with antibody to tyrosine hydroxylase (TH). Nuclei are highlighted with DAPI (4′,6-diamidino-2-phenylindole) in blue. Left: representative images. Right: average number of puncta per cell in >50 TH-positive cells. Scale bars, 10 μm. All values are mean + s.e.m.; differences with Ctr (*) and between WT LRRK2 and G/S LRRK2 (§) were significant at P 0.05; unpaired t-test in a and ANOVA and Bonferroni test in b,c.