EMBO/soda-nel huggingface dataset:
PAX6D Instructs Neural Retinal Specification from Human Embryonic Stem Cell-Derived Neuroectoderm (2020)
Figure 3

Figure 3. Generation of PAX6 isoform specific knockouts and their effects on NR differentiation

Schematic diagram for generating PAX6D knockout (PAX6D KO). The exon α was replaced by PGK-Puromycin. Primers indicated in this panel are used to detect the PAX6D KO.

qPCR verification of PAX6D KO efficiency. PAX6A&B and PAX6D isoforms were also evaluated by qPCR with isoform specific primers (n=3 biological replicates for each group).

Western blot detection of PAX6 isoforms in wildtype and PAX6D KO cells.

qPCR quantification of retinal gene VSX2 in PAX6D KO cells (n=3 biological replicates for each group).

qPCR quantification of retinal gene SIX6 in PAX6D KO cells (n=3 biological replicates for each group).

Immunostaining of VSX2 in wildtype and PAX6D KO cells. Scale bars:100 µm. Samples were from day 30 differentiating retinal cells.

GO analysis of differentially expressed genes in PAX6 KO cells. Up-regulated genes and down-regulated genes are analyzed separately. The biological processes are ranked by p-value. Only top 15 terms were shown.

GO analysis of differentially expressed genes in PAX6D KO cells. Up-regulated genes and down-regulated genes are analyzed separately. The biological processes are ranked by p-value. Only top 15 terms were shown.

Venn diagram shows the overlap of differentially regulated genes in PAX6 KO and PAX6D KO cells. The numbers indicate the number of genes in each area. Also see Figure EV3C for detailed GO analysis of genes unique in PAX6 KO cells.

GO analysis of biological process for overlap genes in panel I. The numbers in the cycle are the ratio of genes. The GO terms are ranked by p-value in the clockwise direction.

Data information: In (B, D, E), data are presented as mean ± SD. Significance was assessed by unpaired Student's t‐test; ***P < 0.001.
PAX6D Instructs Neural Retinal Specification from Human Embryonic Stem Cell-Derived Neuroectoderm (2020)
Figure 4

Figure 4. PAX6D is required for NR specification

Immunostaining of SOX1 and SOX2 in human fetal forebrain and retinal tissues. Scale bars:50 µm.

Flow cytometry analysis of day 7 cell cultures under retinal differentiation from wildtype, PAX6 KO and PAX6D KO cell lines.

Flow cytometry analysis of day 20 cell cultures under retinal differentiation from wildtype, PAX6 KO and PAX6D KO cell lines.

Quantification of SOX1-/SOX2+ ratio in panel C (n=3 biological replicates for each sample).

qPCR quantification of SOX1 in wildtype, PAX6 KO and PAX6D KO cell at day 20 of retinal differentiation (n=3 biological replicates for each sample).

Data information: In (D, E), data are presented as mean ± SD. Significance was assessed by unpaired Student's t‐test; ***P < 0.001.
PAX6D Instructs Neural Retinal Specification from Human Embryonic Stem Cell-Derived Neuroectoderm (2020)
Figure 5

Figure 5. Effects of PAX6A and PAX6D on RPC differentiation

Schematic diagram for generating PAX6A (PAX6A TetOn in PAX6KO) and PAX6D (PAX6D TetOn in PAX6 KO) inducible cell lines in PAX6 KO hESCs. With this design, PAX6A or PAX6D is induced separately with the doxycycline (DOX) treatment.

qPCR quantification of PAX6A and PAX6D gene expression under the treatment of DOX (n=3 biological replicates for each sample).

Western blot analysis of PAX6A and PAX6D protein expression under the treatment of DOX.

Schematic diagram of DOX treatment in PAX6A and PAX6D TetOn cell lines for (E,F,G).

qPCR quantification of SOX1 gene expression in the PAX6A TetOn and PAX6D TetOn cells (n=3 biological replicates for each group).

qPCR quantification of retinal marker expression in PAX6D TetOn cells w/o DOX treatment (n=3 biological replicates for each group).

Cell population composition analysis with SOX1 and SOX2 staining from flow cytometry. SOX1/SOX2 double positive cells represent the neuroepithelia fate while the SOX1 negative and SOX2 positive cells represent the neuroretina fate.

Data information: In (B, E, F), data are presented as mean ± SD. Significance was assessed by unpaired Student's t‐test; *P < 0.05, ***P < 0.001.
PAX6D Instructs Neural Retinal Specification from Human Embryonic Stem Cell-Derived Neuroectoderm (2020)
Figure 6

Figure 6.

Identification of PAX6D potential targets by ChIP-seq

Experimental design for the ChIP samples. PAX6A TetOn and PAX6D TetOn cells were differentiated towards the retinal lineage. At day 6 the cells were treated with DOX to turn on PAX6A or PAX6D expression. After 3 days of treatment, the cells were collected at day 10 and used for ChIP experiment.

The fragment enrichment evaluated by SES plot.

GO analysis of the PAX6A potential targets and PAX6D potential targets identified by ChIP-Seq. PAX6A only, PAX6A and PAX6D shared, and PAX6D only targets are analyzed separately.

Verification of PAX6D target genes using PAX6D TetOn cells. Two sets of genes (retinal and non-retinal neural genes) are directly regulated by PAX6D in opposite directions (n=3 biological replicates for each group).

Data information: In (D), data are presented as mean ± SD. Significance was assessed by unpaired Student's t‐test; *P < 0.05, **P < 0.01, ***P < 0.001.
PAX6D Instructs Neural Retinal Specification from Human Embryonic Stem Cell-Derived Neuroectoderm (2020)
Figure 7

Figure 7. NR specification regulated by PAX6D is WNT dependent

qPCR quantification of WNT8B in PAX6D KO cells at multiple time points during retinal differentiation (n=3 biological replicates for each group).

qPCR quantification of retinal gene expression under the effect of WNT agonist CHIR99021 and WNT antagonist IWR1 on retinal differentiation (n=3 biological replicates for each group).

qPCR quantification of retinal gene expression in PAX6D KO cells treated with WNT antagonist IWR1 to rescue the retinal differentiation (n=3 biological replicates for each group).

qPCR quantification of retinal gene expression in PAX6D TetOn cells when PAX6D was induced by DOX in the presence of WNT agonist CHIR99021 (n=3 biological replicates for each group).

Western blot of WNT8B and ACTIN in PAX6D KO cells with treatment of untreat, control shRNA lentivirus, WNT8B shRNA lentivirus.

qPCR quantification of retinal marker expression in PAX6D KO cells treated with control shRNA lentivirus and WNT8B shRNA lentivirus (n=3 biological replicates for each group).

A model describing the role of a unique function for PAX6D isoform in regulating retinal specification. retinal fate choice versus forebrain is controlled by PAX6D by regulating a set of neural genes and retinal genes.

Data information: In (A-D, F), data are presented as mean ± SD. Significance was assessed by unpaired Student's t‐test; *P < 0.05, **P < 0.01, ***P < 0.001.

Expanded View Figure Legends
Mitophagy receptor FUNDC1 is regulated by PGC-1α/NRF1 to fine tune mitochondrial homeostasis (2020)
Figure 1

Figure 1 Expression of FUNDC1 is induced by cold exposure in BAT

(A-E) BATs were isolated from cold-exposed mice (4℃, 72 h) or control mice (30℃, 72 h), then lysed and subjected to western blotting for analysis of the indicated proteins (A). Relative protein expression levels, normalized to Actin, are shown in (B). mRNA expression levels of the indicated genes were assayed by real-time PCR and normalized to Actb (C). Mitochondrial DNA copy numbers were determined by real-time PCR and normalized to nuclear DNA standards (D). Relative increase of mitochondrial proteins was normalized to mtDNA copy numbers and plotted (E).

Data information: n=6 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using two-tailed Student's t-test. ns: no significant difference; *P<0.05; **P<0.01; ***P<0.001.

(F-G) Representative images showing immunohistochemical detection of FUNDC1 in sections of BATs from mice kept at 30℃ or 4℃ for 72 h. Scale bar, 50 μm. The expression of FUNDC1 was quantified by analyzing and normalizing the average optical density of FUNDC1 staining.

Data information: n=4 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using two-tailed Student's t-test. **P<0.01.
Mitophagy receptor FUNDC1 is regulated by PGC-1α/NRF1 to fine tune mitochondrial homeostasis (2020)
Figure 2

Figure 2 PGC-1α regulates FUNDC1 expression

(A-C) Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30℃ (Normal) or following 72 h at 4℃ (Cold) (A). Relative protein expression levels were normalized to Actin (B). mRNA expression levels of the indicated genes were assayed by real-time PCR and normalized to Actb (C).

Data information: n=6 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using two-tailed Student's t-test. ns: no significant difference; *P<0.05; **P<0.01.

(D-F) Flag-PGC-1α was introduced into PGC-1α knockdown brown preadipocytes for 24 h and samples were collected for western blotting analyses of the indicated proteins (D). Expression levels of the indicated proteins were quantified (E) (mean ± SEM; n=3). Scramble, scramble shRNA. Samples were collected for real-time PCR analysis. The expression of Fundc1 was normalized (F).

Data information: experiments were repeated 3 times, data are represented as the mean±SEM. Statistical analysis was performed using two-tailed Student's t-test. ns: no significant difference;*P< 0.05; **P<0.01.

(G-H) Scramble and PGC-1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses (G) and quantification of the indicated proteins (H)

Data information: experiments were repeated 3 times, data are represented as the mean±SEM. Statistical analysis was performed using two-tailed Student's t-test. ns: no significant difference;**P<0.01.
Mitophagy receptor FUNDC1 is regulated by PGC-1α/NRF1 to fine tune mitochondrial homeostasis (2020)
Figure 3

Figure 3 NRF1 transcriptionally regulates FUNDC1 expression

(A) Schematic representation of human Fundc1 promoter deletion constructs and 2 putative NRF1 binding sites were marked in blue and red and the corresponding luciferase activity expressed as the activity relative to the 2000 bp promoter.

Data information: experiments were repeated 3 times, data are represented as the mean±SEM.

(B) Luciferase activity stimulated by the human Fundc1 promoter. The activity of Fundc1 promoter fragments, either wild type (WT) or with a mutation in the potential NRF1 binding site, was assayed using a dual luciferase reporter system.

(C,D) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 (F1 probe or mut F1 probe) ,the confirmed NRF1 binding site in the promoter of VSNL1 (positive probe). The probes were incubated with HeLa nuclear extracts, then run on a gel (C). A super-shifted band (asterisk) is detected in the presence of an anti-NRF1 antibody (D).

Labeled probe: biotin labeled wild type Fundc1 probe; Cold probe: wild type Fundc1 probe; Mut probe: NRF1 binding site mutant Fundc1 probe; asterisk indicates the supershifted band generated with antibody to NRF1.

(E,F) ChIP assay to analyze the interaction of NRF1 (E) or PGC-1α (F) proteins with the Fundc1 promoter.

(G) NRF1-myc was introduced into NRF1 shRNA knockdown cells for 24 h and scramble cells were used as control and samples were collected for western blotting with the indicated antibodies.

(H) Scramble and NRF1 shRNA knockdown cells were transfected with vector and PGC-1α-Flag for 24 h and samples were collected for western blotting with the indicated antibodies.

(I) ChIP analysis of the interaction of PGC-1α protein with the Fundc1 promoter in scramble or NRF1 shRNA knockdown cells.
Mitophagy receptor FUNDC1 is regulated by PGC-1α/NRF1 to fine tune mitochondrial homeostasis (2020)
Figure 4

Figure 4 Cold exposure induces mitophagy and the interaction between FUNDC1 and LC3

(A,B) Western blotting analyses of the levels of autophagy-related proteins in BAT from mice at the normal temperature (30℃) or at 4℃ for 72 h (A). Expression of the indicated proteins was analyzed before and after 72 h cold exposure (4℃) (B).

Data information: n=3 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using two-tailed Student's t-test. *P< 0.05; **P<0.01.

(C) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature (30 ℃) or 4℃ for 72 h.

(D,E) Representative transmission electron microscope (TEM) images of BAT cells isolated from mice at the normal temperature (30℃) or 4℃ for 72 h. Scale bar, 1 μm (D).The number of mitophagosomes per 100 mitochondria was counted and normalized (E).

Data information: n=3 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using two-tailed Student's t-test. **P<0.01.

(F,G) BATs from mice exposed to normal conditions (30℃) or cold (4℃) were lysed and immunoprecipitated with antibodies against FUNDC1, LC3B or IgG, then immunoblotted with the indicated antibodies. The ratio between immunoprecipitated LC3B to FUNDC1 or FUNDC1 to LC3B were quantified and plotted (G).

Data information: experiments were repeated 3 times, data are represented as the mean±SEM. Statistical analysis was performed using two-tailed Student's t-test. *P<0.05.
Mitophagy receptor FUNDC1 is regulated by PGC-1α/NRF1 to fine tune mitochondrial homeostasis (2020)
Figure 5

Figure 5 FUNDC1 is required for cold-induced mitophagy in mouse BAT

(A,B) BATs isolated from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) mice at 30℃ or following 72 h at 4℃ were analyzed by transmission electron microscopy. Representative images are shown, scale bar, 1μm (A). The number of mitophagosomes per 100 mitochondria were counted and normalized (B).

Data information: n=3 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using two-way ANOVA test. ns: no significant difference; *P<0.05.

(C,D).Representative fluorescence images of BAT sections from Fundc1fl/fl/Adiponectincre- (Cre-) and Fundc1fl/fl/Adiponectincre+ (Cre+) mito-Keima mice at normal conditions (30℃) or 4℃ for 72 h. Scale bar, 50 μm (C). Quantification of the ratio between red and green signals (D).

Data information: n=6 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using two-way ANOVA test. ns: no significant difference; *P<0.05.

(E) Fundcfl/fl/Ucp1cre- (Cre-) and Fundcfl/fl/Ucpcre+ (Cre+) mice were maintained at 30℃ (Normal) or exposed to 4℃ for 72 h (Cold). Mitochondrial DNA copy numbers were analyzed by real-time PCR and normalized to nuclear DNA standards.

(F) The relative total mito-keima signal (green signal ) in Fundc1 deficiency and control mito-keima mice under normal condition or cold exposure were quantified and plotted.

Data information: n=6 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using two-way ANOVA test. ns: no significant difference; **P<0.01.

(G) BATs isolated from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) at 30℃ or following 72 h at 4℃ were analyzed by transmission electron microscopy. The percent of mitochondria without cristae is shown in the histogram.

(H,I). Oxygen consumption rate (OCR) (H) and mitochondrial superoxide (I) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.

Data information: n=3 or 4 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using two-way ANOVA test. ns: no significant difference; *P<0.05
Mitophagy receptor FUNDC1 is regulated by PGC-1α/NRF1 to fine tune mitochondrial homeostasis (2020)
Figure 6

Figure 6 FUNDC1 regulates adaptive thermogenesis and contributes to cold tolerance

(A) Body temperature measurements in Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) fasting male mice before and during cold exposure (4°C) for the indicated time.

Data information: n=6 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using repeated measures ANOVA. ***P<0.001

(B,C) Body temperature (B) and BAT tissue weight (C) measurements before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice.

Data information: n=6 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using repeated measures ANOVA or two-way ANOVA test. ns: no significant difference; * P<0.05; ***P<0.001.

(D-F) Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Western blotting analysis of BAT protein levels (D). Protein expression levels were normalized to Actin (E). Levels of mRNAs encoding Fundc1 and Ucp1 were measured by real-time PCR and normalized to Actb (F).

Data information: n=6 biological replicates, data are represented as the mean±SEM. Statistical analysis was performed using two-way ANOVA test. * P<0.05; **P<0.01; ***P<0.001.

(G) Representative images of UCP1 immunohistochemistry of BAT sections from Cre- or Cre+ mice in normal conditions (30℃) or following 72 h at 4℃. Scale bar, 50 μm.

(H) Schematic model of PGC-1α-NRF1-FUNDC1 axis in mitochondrial quality and quantity control and adaptive thermogenesis.
Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt (2016)
Figure 1

Figure 1. Loss of CLPP alleviates cardiomyopathy in DARS2-deficient heart.

(A) Kaplan-Meier survival curves (n = 12 - 16). (B) Heart gross morphology. (C) Body weight; (D) Heart weight and (E) Heart-to-body weight ratio (n = 28-40). (F) Relative expression levels of cardiac hypertrophy markers (Nppa and Nppb) (n = 5). Bars represent mean ± S.E.M. (Student’s t test; **p < 0.01, ****p < 0.0001). (G) Enzyme histochemical double staining for COX and SDH activities. (n = 4). (H) Assessment of cardiac fibrosis by Masson’s trichrome staining (n = 4). White scale bars, 100μm; black scale bars, 1 mm.
Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt (2016)
Figure 2

Figure 2. Mitochondrial and cellular stress responses do not depend on CLPP.

(A) Western blot analysis and (B) Relative quantification of UPRmt markers in heart extracts. HSC70 is used as a loading control (CTRL), (n = 3). (C and D) Relative expression levels normalized to WT of (C) established UPRmt markers and (D) mitochondrial chaperones, proteases and TFAM in 3 – 4 week old mice (n = 3 – 4). (E) Western blot analysis of cellular stress markers. HSC70 is used as a loading control (CTRL), (n = 3). (F) Relative expression levels of transcription factors involved in UPRmt and ISR (left) and Fgf21 (right) (n = 5). Bars represent mean ± S.E.M. (Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt (2016)
Figure 4

Figure 4. Dysregulation of mitochondrial protein synthesis is partially rescued by the loss of CLPP.

(A) Representative gel of the In organello translation analysis of heart mitochondria. De novo synthetized proteins are isolated after labeling with 35S-met (1 hour Pulse) or after cold chase (3h Chase). Positions of individual proteins are indicated on the left. Position of full-length proteins (Proficient) and low molecular weight polypeptides (Abortive) are indicated on the right. Note that in WT and ClpP KO low molecular weight polypeptides include ATP8/ND4L proteins. (B) Relative overall protein synthesis and turnover rate. (C) Western blot analysis of DARS2 levels. HSC70 is used as a loading control (CTRL). (D) Relative levels of the individual de novo synthesized OXPHOS subunits in DKO mitochondria normalized to the corresponding Dars2 KO polypeptide level. (E) Relative levels of protein synthesis of Proficient and Abortive polypeptides. (F) Quantification of the turnover rate of full-length (Proficient) de novo synthesized proteins. Bars represent mean ± S.E.M. (Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), (n=3-4).
Early auto-immune targeting of photoreceptor ribbon synapses in mouse models of multiple sclerosis (2018)
Figure 1

Figure 1. CASPR1 and CNTN1 co-immunoprecipitate with RIBEYE. (A) Coomassie- stained proteins from bovine retina that were co-immunoprecipitated by the anti-RIBEYE antibody 2D9 (lane 3). Lane 2, control precipitation by irrelevant mouse IgGs; lane 1, input (10% of the total loaded in each lane). (B) Western blot of the immunoprecipitated proteins (as in A, 1% of the total protein was loaded per lane) probed with anti-RIBEYE (Ba), anti-CASPR1 (Bb) and anti-CNTN1 (Bc). The 2D9 antibody immunoprecipitated RIBEYE (Ba, lane 3), CASPR1 (Bb, lane 3) and CNTN1 (Bc, lane 3); these proteins were not detectable in the control immunoprecipitations. Bd) As a loading control, heavy chains of the precipitating antibody (IgG hc) were visualized by the goat anti-mouse IgG antibody conjugated to peroxidase.
Early auto-immune targeting of photoreceptor ribbon synapses in mouse models of multiple sclerosis (2018)
Figure 2

Figure 2. Localization of CASPR1 and visual-arrestin (v-arrestin) in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelled with antibodies against CASPR1 and RIBEYE (A-B) or with antibodies against v-arrestin and RIBEYE (C-D). For quantification of the immunolabelling data, please see Fig. 3C. V-arrestin, visual arrestin; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μm.
Early auto-immune targeting of photoreceptor ribbon synapses in mouse models of multiple sclerosis (2018)
Figure 3

Figure 3. Localization of CNTN1 in semi-thin (0.5μm-thin) sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). (A,B) Sections were double-immunolabelled with antibodies against CNTN1 and RIBEYE. (C) Quantification of the immunosignals of the indicated proteins in the OPL and IPL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer; n.s., non-significant; ***, p<0.001 (Mann-Whitney U test, actual p-values are given in C) (N=6 independent set of experiments; n=295 images (CFA); n=274 images (MOG/CFA) for quantification of RIBEYE immunosignals; n=95 (CFA); n=92 (MOG/CFA) for quantification of CASPR1 immunosignals; n=91 (CFA), n=82 (MOG/CFA) for CNTN1 quantification; n=109 (CFA); n=100 (MOG/CFA) for quantification of v-arrestin. Scale bars: 20μm.