EMBO/soda-nel huggingface dataset:
Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα (2020)
Figure 1

Figure 1. KDF1 associates with IKKα.

(A) Tandem affinity purification was used to isolate KDF1 associated proteins from SILAC-labelled keratinocytes. Precipitated proteins were resolved by SDS-PAGE and subjected to identification with LC-MS/MS. Arrows denote IgG heavy and light chains. Star denotes KDF1. Ctrl: control. Kd: kilodalton for molecular weight markers.

(B-C) HEK293 cells were transfected with empty vector or plasmids encoding HA tagged KDF1 and/or GST-tagged IKKα. Immunoprecipitation (α-HA or α-GST) was carried out to determine their interaction. Immunoprecipitates (IP) and whole cell lysate (WCL) were immunoblotted (IB) with different antibodies as indicated.

(D) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca). IPs were blotted with different antibodies as indicated. Star denotes KDF1. The same IP was used for USP7 immunoblot as shown in Fig. 5B.

(E) HEK293 cells were transfected to co-express HA-tagged KDF1 with GST-tagged IKKα or its kinase dead (KD) mutant. IP and WCL were blotted with different antibodies as indicated.

(F) HEK293 cells were transfected to co-express HA-tagged KDF1 with GST-tagged IKKα or its different truncation mutants (EV Fig. 1B). IP and WCL were blotted with different antibodies as indicated. Arrow denotes antibody heavy chain.

(G) HEK293 cells were transfected to co-express GST-tagged IKKα with HA-tagged KDF1 or its different truncation mutants (EV Fig. 1C). IP and WCL were blotted with different antibodies as indicated.
Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα (2020)
Figure 2

Figure 2. Loss of KDF1 or IKKα leads to aberrant epidermal differentiation.

(A) H/E staining of E18.5 skin sections from WT, IKKα KO, and KDF1 KO mice. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=50 μm.

(B) E18.5 skin sections from WT, IKKα KO, and KDF1 KO mice were immunostained with different antibodies as indicated (Krt14: Keratin 14, Krt10: Keratin 10, Lor: Loricrin, β4: β4-integrin, CD104). Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=100 μm.

(C) Immunoblots of WCL collected at 0, 1 and 2 days after calcium shift with different antibodies as indicated.
Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα (2020)
Figure 3

Figure 3. IKKα and KDF1 interaction is essential for skin stratification.

(A) Immunoblots of WCL from IKKα KO cells with rescued expression of HA-tagged IKKα or its mutant using different antibodies as indicated.

(B) Immunoblots of WCL from KDF1 KO cells with rescued expression of KDF1 or its mutant using different antibodies as indicated. Expression of exogenous KDF1 was induced by doxycycline (DOX) at different concentration.

(C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05

(E) Sections of regenerated skin developed from engrafted IKKα KO, KDF1 KO, and their rescued cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=50 μm.
Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα (2020)
Figure 4

Figure 4. IKKα protein level is crucial during keratinocyte differentiation and KDF1 regulates the ubiquitination and the protein level of IKKα.

(A) WCL from WT and KDF1 KO cells before and after calcium shift were immunoblotted with different antibodies as indicated. Lo: low calcium; Hi: high calcium.

(B-C) WT and KDF1 KO keratinocyte were treated with 20nM cycloheximide (CHX). WCL was collected at 0, 30, 60, 90, 120 min post CHX treatment and subjected to immunoblotting with IKKα antibody (B). Band intensity is determined by densitometry and the amount of IKKα is calculated and quantified (C). Statistical analysis is conducted using 2-way ANOVA. N=3 (biological replicates). *, p<0.05. Error bar represents S.D. (standard deviation).

(D-E) WT and KDF1 KO keratinocyte (right panels) or cells transfected with plasmid encoding exogenous IKKα (left panels) were treated with MG132 at 10µM for 6 hours, then were subjected to immunoprecipitation using anti-ubiquitin (Ub) antibody. IP and WCL were analyzed by immunoblots with α-IKKα antibody (D). Overall intensity of all ubiquitinated IKKα bands was determined by densitometry. Ratio of ubiquitinated IKKα was quantified and presented as bar graphs (E). Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). *, p<0.05. Kd: kilodalton for molecular weight markers.

(F) IKKα protein level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant, under both low and high calcium conditions, was examined and quantified by western blotting (quantification shown in EV Fig. 4C).

(G) PiggyBac transposon was used to ectopically express HA-tagged IKKα in KDF1 deficient cells. WCL were collected and analyzed by immunoblots with different antibodies as indicated.

(H) Overexpression of IKKα in KDF1 KO keratinocyte can restore normal skin stratification. Skin sections from grafted tissue were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bars = 50 µm.
Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα (2020)
Figure 5

Figure 5. USP7 regulates IKKα ubiquitination and skin differentiation.

(A) HEK293 cells were transfected to co-express KDF1 with USP7. IP and WCL were blotted with different antibodies as indicated.

(B) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca), same as in Fig. 1D. IPs were blotted with different antibodies as indicated. The star denotes KDF1.

(C) WCL from WT and USP7 CRISPR KO cells were subjected to immunoblotting with different antibodies as indicated.

(D) IKKα protein level in WT and USP7 KO keratinocyte were examined by immunoblotting before and after calcium shift.

(E) WT and USP7 KO keratinocyte were treated with MG132 at 10μM for 6 hours, then subjected to immunoprecipitation using anti-ubiquitin antibody. Precipitated product was analyzed by immunoblotting with IKKα antibody. Band intensity was determined by densitometry and shown as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). **, p<0.01. Kd: kilodalton for molecular weight markers.

(F-G) Immunoblot of Krt10 and Loricrin (Lor) with WT and USP7 KO cells before and after calcium shift (F). Star denotes an unspecific band in α-Loricrin blots. Band intensity was determined by densitometry and shown as bar graphs (G). Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ****, p<0.0001; **, p<0.01.

(H) Sections of engrafted skin developed from USP7 KO or control WT cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=100μm.

(I) A working model of epidermal differentiation regulated by KDF1. Upon differentiation signals, KDF1 associates with IKKα and recruits deubiquitination enzyme USP7 to the protein complex. USP7 can deubiquitinate IKKα and promote its protein stability, which will in turn promote epidermal differentiation.

Expanded view Figure Legends
The Plant FYVE Domain-containing Protein FREE1 Associates with Microprocessor Components to repress miRNA Biogenesis (2022)
Figure 1

Figure 1. FREE1 associates with the core microprocessor

A. Schematic domain structure of FREE1 and CPL1 protein. P-rich: prolinerich region, FYVE: FYVE domain, CC: coiled coil domain, dsRBM: dsRNA binding motif.

B. Yeast two-hybrid analysis of the interactions between FREE1 and core microprocessor. SD-2: SD/-Leu-Trp. SD-4: SD/-Ade-His-Leu-Trp. Three dots represent three yeast colonies randomly picked out in each sample.

C. BiFC assay to verify the interaction between FREE1 and CPL1. YC-FREE1 and YN-CPL1, were co-expressed with mCherry-HYL1, SE-mCherry, or DCL1-mCherry in Arabidopsis protoplasts, respectively, followed by confocal observation. Scale bar, 5 μm.

D. Co-immunoprecipitation analysis of interactions between FREE1 and CPL1 and CPL2. Arabidopsis protoplasts co-expressing mCherry-FREE1 with YFP, YFP-CPL1 or YFP-CPL2 were subjected to protein extraction and IP with GFP-trap followed by immunoblotting with mCherry and GFP antibodies.

E. Co-immunoprecipitation analysis of the associations between FREE1 and microprocessor components. Transgenic plants expressing GFP-FREE1 were subjected to protein extraction and IP with GFP-trap followed by immunoblotting with indicated antibodies.
The Plant FYVE Domain-containing Protein FREE1 Associates with Microprocessor Components to repress miRNA Biogenesis (2022)
Figure 3

Figure 3. FREE1 affects the phosphorylation of HYL1

A. Detection of HYL1 phosphorylation in Col-0, free1-ctmut or cpl1-8. Proteins extracted from 7-day-old Arabidopsis seedlings were separated by Phos-tag polyacrylamide gels or SDS polyacrylamide gels followed by immunoblotting with indicated antibodies. The numbers in the first row indicate the relative grayscale intensities of total HYL1, the numbers in the second row indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1, and the intensity in WT was arbitrarily set to 1.00.

B. Transgenic plants expressing pHYL1:: YFP-HYL1 in Col-0, free1-ctmut or cpl1-8 backgrounds were subjected to protein extraction and IP with GFP-trap, followed by detection with phos-tag BTL-111 or GFP antibody. The numbers indicate the relative intensities of each band detected with phos-tag BTL-111 and anti-GFP, and the first lane was arbitrarily set to 1.00.

C. Confocal observation of the YFP-HYL1-containing D-bodies in Col-0 and free1-ctmut roots. Arrows indicated the YFP-HYL1-containing D-bodies. Scale bar, 5 μm.

D. Statistically analysis of the number of YFP-HYL1-containing D-bodies as shown in Fig 4C. The X-axis represents the number of D-bodies per cell, and the Y-axis represents the percentage of cells with the corresponding numbers. The quantification was performed by observing more than 400 cells from 20 roots for each genotype, data are presented as means ± s.d. The asterisk indicates significant difference between the samples (**P < 0.01 by Student's t-test, compared with WT).

E. Protein abundance of core components of miRNA processing complex in Col-0 and free1-ctmut. Proteins extracted from 7-day-old Arabidopsis seedlings were used for immunoblotting with indicated antibodies. The numbers represent the intensities of each band detected with indicated antibody relative to anti-UGPase, and the first lane was arbitrarily set to 1.00.

F. Proteins extracted from 7-day-old Arabidopsis seedlings treated with CHX (0.5 mM) in dark for 0h, 2h, 4h were used for immunoblotting analysis with indicated antibodies. The numbers represent the relative intensities of each band detected with anti-HYL1 and anti-Actin, and the first lane was arbitrarily set to 1.00.

G. Proteins extracted from 7-day-old Arabidopsis seedlings with 3-d continuous light or dark treatment were used for immunoblotting analysis with indicated antibodies.
The Plant FYVE Domain-containing Protein FREE1 Associates with Microprocessor Components to repress miRNA Biogenesis (2022)
Figure 4

Figure 4. FREE1 affects the HYL1-SE-CPL1 association and functions genetically upstream of CPL1 in affecting HYL1 phosphorylation

A. Proteins extracted from Arabidopsis protoplasts co-expressing YFP-CPL1 and different amount of 3myc-FREE1 were used for immunoprecipitation with GFP-Trap followed by detection with indicated antibodies.

B. Proteins extracted from transgenic plants expressing YFP-CPL1 in Col-0 or free1-ctmut background were used for IP with GFP-trap followed by immunoblotting analysis with indicated antibodies.

C. Statistically analysis of the relative intensity of FREE1, HYL1 and SE in Col-0 or free1-ctmut background (IP/Input). Data are presented as means ± s.d. (3 biological replicates), the asterisk indicates significant difference between the samples (**P < 0.01 by Student's t-test, compared with WT).

D. Transgenic plants of YFP-HYL1 / WT, YFP-HYL1 / free1-ctmut, YFP-HYL1 / cpl1-8 and GFP / WT were subjected to protein extraction and IP with GFP-trap followed by immunoblotting analysis with indicated antibodies.

E. Statistically analyzed the relative intensity of FREE1 and HYL1(IP/Input). Data are presented as means ± s.d. (3 biological replicates), the asterisk indicates significant difference between the samples (**P < 0.01 by Student's t-test, compared with WT).

F. Proteins extracted from 7-day-old Arabidopsis seedlings were separated by Phos-tag polyacrylamide gels followed by immunoblotting analysis with HYL1 antibody. The numbers indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1, and the first lane was arbitrarily set to 1.00.
miR181a/b down regulation exerts a protective function on mitochondrial disease models (2019)
Figure 1

Figure 1. miR-181a/b silencing increases mitochondrial biogenesis and mitophagy and protects from FCCP-induced cell death. A) q-RT-PCR reveals that miR-181a/b silencing leads to upregulation of miR-181a/b predicted targets in SH-SY5Y cells. N=3 independent experiments. B) miR-181-mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3'-UTR predicted target sequences. Point mutations (mut) in miR-181a/b binding sites abolish Luciferase repression in all cases apart from PPARGC1A. Data are normalized to negative mimic transfection (dashed line). N=6 independent experiments. C) q-RT-PCR reveals upregulation of miR-181a/b targets in the eyes of miR-181a/b-1-/- vs. miR-181a/b-1+/+ animals. n≥5 animals/genotype. D) miR-181a/b-1-/- mice show increased mtDNA content vs. miR-181a/b-1+/+ mice as measured by q-PCR. N=4 animals/genotype. E) WB analysis (left panel) reveals increased levels of mitochondrial proteins in the eyes of miR-181a/b-1-/- (-/-) vs. miR-181a/b-1+/+ (+/+) mice (quantified in the right panel). Data are normalized to either p115 or Gapdh. N≥3 animals/genotype. Please note that all compared bands from +/+ and -/- samples are from the same blots which were cropped and shown split for the sake of data presentation clarity. F) WB analysis on mitochondrial and cytosolic fractions (left panel) show increased levels of p62 and Parkin in mitochondrial fraction from the eye of -/- vs. +/+ mice (quantified in the right panel). Data are normalized to Ndufb8 or Gapdh for mitochondrial and cytosolic fractions, respectively. N=3 animals/genotype. G) Cell death analysis shows that miR-181a/b silencing protects SH-SY5Y cells from FCCP treatment. N≥7 independent experiments.

p‐values were calculated by one-tailed Student's t-test in A-C and in E, by two-tailed Student's t-test in D and by two-way ANOVA with post-hoc Tukey analysis in G; Error bars are SEM.
miR181a/b down regulation exerts a protective function on mitochondrial disease models (2019)
Figure 2

Figure 2. miR-181a/b inhibition counteracts cell death in MLS medakafish models. A) TUNEL assays on eye and brain sections of stage (st)30 medakafish control and MO-injected embryos reveal a decrease in the number of apoptotic cells in both hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected compared to hccs-MO- and cox7b-MO-injected embryos. Scale bars are 20μm. B) TUNEL-positive cells/eye. n≥5 eyes for each model. C) Caspase assays show restored levels of caspase-3 and caspase-9 activities in hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected embryos with respect to hccs-MO- and cox7b-MO-injected embryos. n≥5 embryos for each model.

p‐values were calculated by Analysis of Deviance for Negative Binomial Generalized Linear Model in B and by one-way ANOVA with post-hoc Tukey analysis in C; Error bars are SEM.
miR181a/b down regulation exerts a protective function on mitochondrial disease models (2019)
Figure 3

Figure 3. miR-181a/b downregulation ameliorates the phenotype of MLS medakafish models.

(A-I) Representative images of st30 medaka embryos injected with hccs-MO (B) and cox7b-MO (H) alone or co-injected with miR-181a/b-MOs (C,I). Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in both hccs-MO and cox7b-MO embryos. (D-F, J-L) hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in both hccs-MO/miR-181a/b-MO and cox7b-MO/miR-181a/b-MO embryos (D, J). PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation (E, K). HA14-1 treatment counteracts the effect of miR-181a/b downregulation in the hccs-MO/miR-181a/b-MO model but not in the cox7b-MO/miR-181a/b-MO embryos (F, L). Scale bars are 100μm. M-N) Percentage of embryos with or without MLS phenotype in the different conditions illustrated in (A-L) in hccs-MO (M) and cox7b-MO (N) embryos. N≥300 embryos/conditions.

p‐values were calculated by Analysis of Deviance for Generalized Linear Model; Error bars are SEM.
miR181a/b down regulation exerts a protective function on mitochondrial disease models (2019)
Figure 4

Figure 4. Inactivation of miR-181a/b protects RGCs from cell death in a rotenone-induced mouse model of LHON syndrome. A) Immunofluorescence analysis with anti-NeuN antibody in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. RGCs are preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. Scale bars are 50μm. B) Number of RGCs/mm (indicated as %) at one- and two-weeks post-injection. N=10. C) NADH dehydrogenase histochemical reaction on retinal sections of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. At one week post-injection, NADH dehydrogenase activity is lost in RGCs (GCL, areas within dashed lines) of miR-181a/b-1+/+ Rotenone-injected eyes, while it is preserved in those of miR-181a/b-1-/- Rotenone-injected eyes. GCL, Ganglion Cells Layer ONL; INL, Inner Nuclear Layer; Outer Nuclear Layer. Scale bars are 50μm. D) Immunofluorescence analysis, with an anti-CoxIV antibody, in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice, injected with either Rotenone or DMSO, showed preserved mitochondria in miR-181a/b-1-/- rotenone-injected eyes at one-week post-injection. Dashed boxes indicate the area of magnifications shown in right panels. Scale bars are 10μm. E) Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. N=10.

p‐values were calculated by one-way ANOVA with post-hoc Tukey analysis; Error bars are SEM.
miR181a/b down regulation exerts a protective function on mitochondrial disease models (2019)
Figure 5

Figure 5. miR-181a/b depletion protects RGCs and ameliorates visual function in Ndufs4-/- mice.

A-D) Immunofluorescence analysis with anti-NeuN antibody in the retina of WT, Ndufs4-/-, Ndufs4-/-/miR-181a/b-1-/- and miR-181a/b-1-/- mice. RGCs are preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. Scale bars are 50μm. E) Number of RGCs/mm (%). N=5. F) Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. N≥8. G, H) Electroretinographic analysis reveals amelioration of b-wave patterns in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/-mice. N≥5.

p‐values were calculated by one-way ANOVA with post-hoc Tukey analysis in E-F, and by Two-ANOVA Repetaed Measures with post-hoc analysis in G. p‐values in G refers to 20 log cd.s/m2 and Photopic points. p‐values for the other points are reported in the Appendix Table S1; Error bars are SEM.
miR181a/b down regulation exerts a protective function on mitochondrial disease models (2019)
Figure 6

Figure 6. miR-181a/b depletion enhances mitochondria biogenesis Ndufs4-/- mice. A-C) Electron microscopy analysis shows amelioration of mitochondria morphology and increase of mitochondrial number in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. Scale bars are 1μm. The quantitative increase of mitochondria is reported in C as number of mitochondria/RGC, at p30 and p55. N≥2 animals/genotype. D) Ndufs4-/-/miR-181a/b-1-/- mice show increased mtDNA content vs. Ndufs4-/- mice as measured by q-PCR. N≥4 animals/genotype. E) Biochemical activity of MRC complexes I, II and IV normalized by the percentage of citrate synthase (CS) activity in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. N≥3 animals/genotype. p‐values were calculated by two tailed Student's t-test; Error bars are SEM.
CSAG2 is a cancer-specific activator of SIRT1 (2020)
Figure 1

Figure 1. CSAGs Are Aberrantly Expressed in Cancer and Correlate with Poor Patient Prognosis.

(A) CSAG genes cluster with MAGE-As on chromosome Xq28.

(B) Sequence identity between CSAG genes. Note isoforms of CSAG2/3 are splice variants.

(C-D) RNA-Seq data from GTEx of the normalized expression of human CSAG1 (C) and CSAG2 (D) in the indicated tissues. Number of biological replicates are as follows: testis 361, brain 255, small intestine 187, white adipose 663, muscle 803, skin 604, esophagus 555, stomach 359, liver 226, heart 429. blood vessel 663, lung 578, colon 373, nerve 619, pituitary 283, blood 755, adrenal gland 258, kidney 85, prostate 245, salivary gland 162, ovary 180, breast 459, pancreas 328, vagina 156, uterus 142, spleen 241, fallopian tube 9, bladder 21, cervix 10. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.

(E-F) Expression of CSAG1 (E) and CSAG2 (F) in normal adjacent (blue) or tumor tissue (red) from TCGA RNA-Seq data. N.D. represents normal adjacent is not available. Data visualized by firebrowse. Number of biological replicates are as follows: ACC 41, BLCA 307, BRCA 524, CESC 175, CHOL 11, COAD 153, COADREAD 232, DLBC 36, ESCA 121, HNSC 416, KICH 46, KIPAN 515, KIRC 411, KIRP 58, LAML 7, LGG 528, LUAD 301, LUSC 400, MESO 31, OV 183, PCPG 70, PRAD 204, READ 79, SARC 156, SKCM 422, STAD 288, STES 409, TGCT 126, THCA 408, THYM 75, UCEC 306, and UCS 37. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.

(G) Xq28 MAGE and CSAG genes are frequently co-expressed in lung squamous carcinoma tumors (n=184). RNA-Seq gene expression data analyzed from TCGA. Statistics shown were calculated by linear regression analysis.

(H-K) Expression of CSAG1 (H-I) or CSAG2 (J-K) in patients from indicated cancer types correlates with poor overall survival. Data is from TCGA and visualized by kmplotter.