EMBO/soda-nel huggingface dataset:
TFG binds LC3C to regulate ULK1 localization and autophagosome formation (2021)
Figure 1

Figure 1. TFG interacts with ULK1 and regulates its stability and localization

A) HeLa cells were cultured in complete growth or starvation (STV) media for 30'. Cell lysates were immunoprecipitated (IP) with anti‐TFG antibody or IgG as a negative control. Immunoprecipitated complexes were analysed by WB to detect TFG, ULK1, ATG13, and FIP200 as indicated.

B) HeLa cells were cultured in complete growth (FED) or starvation (STV) media for 1h. Cells were fixed, permeabilized, and labelled with anti-TFG (red) and anti-ULK1 (green) antibodies respectively. Hoechst was used to stain nuclei. Co-localization analysis were performed by ImageJ plugin Jacop and reported in the graph (right). Values of Mander's coefficient for ULK1 are expressed as mean ± SEM. Significance was assigned by unpaired Student's t‐test. **, P<0,01. (n = 3 independent experiments, n=12 fields analysed). Scale bar 5 μm.

C) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) for 48h. Protein extracts were analysed by WB to detect ULK1, TFG and ACTIN as indicated. Densitometry analysis of ULK1 over ACTIN is shown (right). All values are mean ± SEM. Statistical analyses were performed by two‐tailed Student's t‐test. ***, P<0.001. (n=3 independent experiments).

D) HeLa cells were co-transfected with control siRNA and an empty vector (EV), or 3'UTR TFG siRNA together with an EV or HA-TFG plasmids. Protein extracts were analysed by WB to detect ULK1, TFG and ACTIN as indicated. Densitometry analysis of ULK1 protein levels normalized over ACTIN is also reported (right). All values are expressed as the mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by Tukey's multiple comparison test. *, P<0.05; **, P<0.01. (n=3 independent experiments).

E) HeLa cells were transfected as in C), grown in the presence or not of cycloheximide (CHX) for indicated time periods. Protein extracts were analysed by WB to detect ULK1, TFG and ACTIN as indicated. Densitometry analysis of ULK1 normalized over ACTIN is also shown (right). Values are mean ± SEM Statistical analyses were performed by multiple t-test. *, P<0.05. (n=3 independent experiments).

F-G) HeLa cells were treated as in B) and then fixed and stained with ULK1 (green) (F) or ATG13 (red) antibodies (G) respectively. Hoechst was used to stain nuclei. Graphs (right) show the average of both ULK1 (F) (n=3 independent experiments, n=11 fields analysed) or ATG13 (n=3 independent experiments, n≥16 fields analysed) (G) puncta number per cell and both ULK1 (n=3 independent experiments) (F) or ATG13 (n=3 independent experiments) (G) fold of increase of the average size respectively. White arrows indicate most representative puncta of analysed conditions. All data are reported as the mean value ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ****, P<0.0001 or by unpaired Student's t‐test when two groups were compared. *, P<0.05; ****, P<0.0001. Scale bar 5 μm.
TFG binds LC3C to regulate ULK1 localization and autophagosome formation (2021)
Figure 2

Figure 2. TFG is essential for proper ULK1 complex distribution during autophagy

A-D) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed or starved conditions for 1h. Cells were fixed and co-labelled with ULK1 (red) antibody and A) anti-CALNEXIN (CANX) (green) antibody to highlight ER structures; (n=3 independent experiments, n≥16 fields analysed) B) GOLGIN97 (green) antibody to label GOLGI complex; (n=3 independent experiments, n=12 fields analysed) C) SEC31A (green) to point out COPII vesicles. (n=3 independent experiments, n≥16 fields analysed) D) ERGIC53 (green) to mark ERGIC compartment. (n=3 independent experiments, n≥14 fields analysed). Analysis of ULK1 localization with each compartment is reported in each graph. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for ULK1 are expressed in percentage as mean ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ***, P<0,001; ****, P<0,0001; ns, not significant. Scale bars 5 μm. White arrowheads point at ULK1 puncta associated to the analysed markers; N, nucleus.

E) HeLa cells were transfected as in A), labelled with the indicated antibodies and imaged by SIM microscope. Scale bar in large panels, 5 μm; in small panels, 1 μm.

F) HeLa cells were co-transfected with control siRNA and an empty vector (EV), or 3'UTR TFG siRNA together with an EV or HA-TFG plasmids. Cells grown for 1h in starvation medium then fixed, permeabilized, and labelled with anti-ULK1 (green) and anti-ERGIC53 (red) antibodies respectively. Hoechst was used to stain nuclei. Co-localization analysis were performed as in D). Values of Mander's coefficient for ULK1, expressed as percentage, are mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by Dunnetts's multiple comparison test. *, P<0,05. (n=3) (n=3 independent experiments, n≥9 fields analysed). Scale bar 5 μm.
TFG binds LC3C to regulate ULK1 localization and autophagosome formation (2021)
Figure 3

Figure 3. Inhibition of TFG results in impaired autophagy flux

A) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed (FED) or starvation conditions (STV) in the presence or absence of CLQ for 1h. Cells were then fixed and stained using LC3B-II (red) antibody. Analysis of LC3B puncta number is reported (bottom). All data are expressed as the mean value ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. ****, P<0.0001. (n=4 independent experiments, n≥25 fields analysed). Scale bar 5 μm.

B) HeLa cells were transfected as in A) and cultured in starvation conditions (STV) in the presence or absence of Bafilomycin A1 (Baf A1) for 1h. Cells were then fixed and stained using LC3B-II (green) and LAMP1 (red) antibodies. Hoechst was used to mark nuclei.

Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for LC3B-II are expressed as mean ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0,05; (n=3 independent experiments, n≥13 fields analysed). Scale bar 5 μm.

C) HeLa cells were transfected as in A) and grown in fed (FED) or starvation conditions (STV) for 1h. Cells were then fixed and stained using ATG16L1 (yellow) antibody. ATG16L1 puncta number (upper right) and the average size of ATG16L1 as fold increase (bottom right) were reported. All values are expressed as mean ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ****, P<0.0001 or by unpaired Student's t‐test when two groups were compared. ns, not significant. (n=3 independent experiments, n≥9 fields analysed). Scale bar 5 μm.

D) HEK293 cells stably expressing GFP-DFCP1 were transfected as in A) and cultured in starved conditions for 40'. Cells were visualised with cellSens microscope for live-cell imaging. Pictures show the images taken at the same number of frames for each video. Analyses of the number of DFCP1 structures per cell (upper right) and the mean of omegasomes structures diameter (μm) (bottom right) were reported. N, nucleus. All values are expressed as mean ± SEM. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; ***, P<0.001. (n=3 independent experiments). Scale bar 5 μm.
CystatinB is essential for proliferation and interneuron migration in individuals with EPM1 epilepsy (2020)
Figure 1

Figure 1. CSTB expression during cortical development in hCOs and mice

A. CSTB gene expression analysis in hCOs, starting from day (d) 16 until d140. For every time point, at least 3 different samples were analyzed; each sample was made by a pool of 3 to 4 hCOs. Data are represented as mean ± SEM. Unpaired t-test (*P < 0.05, **P < 0.01).

B. Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like).

C. Real time qPCR results of PAX6, NEUN, NESTIN, CSTB gene expression levels in PAX6- and NEUN- FACS sorted nuclei from f-CTRL d135 hCOs. Data are represented as mean ± SEM. Statistical significance was based on T-student test (*P < 0.05, **P < 0.01). For PAX6, 10 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 5 for Neun+sorted Nuclei; for NEUN, NESTIN and CSTB, 9 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 4 for Neun+sorted Nuclei.

D. Western blot analysis and relative quantifications (arbitrary units, a.u.) on protein extracts from cortical (Cx) and ganglionic eminence (GE) tissues for Cstb using Actb as a loading control. Cx and GE tissues from mouse embryos at different time of development as indicated in figure.

E. Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin (acTub) as marker for stabilized microtubules. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.

Nuclei (blue) are stained with DAPI.

Data information: Scale bars: 50 µm in (B); 100 µm in (E); 20 µm in (e', e'' and e''').

Exact p-values in Appendix Table S1.
CystatinB is essential for proliferation and interneuron migration in individuals with EPM1 epilepsy (2020)
Figure 2

Figure 2. CSTB overexpression induces progenitor cells expansion in hCOs and in the developing mouse cortex

A. Micrograph sections of d40 hCOs electroporated with GFP-empty vector control or GFP-CSTB and analyzed 5 dpe. Sections were then immunostained for GFP and KI67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like).

B. Quantification of the total number of proliferating KI67+ cells/area (µm2) of V-like structures transfected with GFP-empty vector control or GFP-CSTB in (A).

C. Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb and analyzed 2 dpe and immunostained with GFP,Ki67 and BrdU 30 minutes after BrdU injection. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.

D,E. Quantification of the total number of proliferating Ki67+ cells/area(µm2)(D) and Ki67+Brdu+ cells/area(µm2)(E) of ventricles transfected with GFP-empty vector or GFP-Cstb in (C).

F,G. Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3)(F) and with GFP, Pax6 and Tbr2 (G). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.

H,I. Distribution of proliferating Ki67+/BrdU+cells (H) and mitotic progenitor PH3+ cells (I) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells or Ki67+/BrdU+ cells calculated in each Bin.

J,K. Distribution of Tbr2+ intermediate progenitors (J) and Pax6+Tbr2+ cells (K) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2+ cells or Pax6+Tbr2+ cells calculated in each Bin.

Data information: Scale bars: 50 μm in (A); 100 μm in (C, F, G). Data are represented as mean ± SEM. Statistical significance was based on Mann-Whitney test (*P < 0.05, **P < 0.01). Every dot in the plots refers to: independent analyzed ventricles per hCO from at least 3 different hCOs generated in at least 2 independent batches, or independent analyzed mouse brains.

Exact p-values in Appendix Table S1.
CystatinB is essential for proliferation and interneuron migration in individuals with EPM1 epilepsy (2020)
Figure 3

Figure 3. CSTB is secreted and induces recruitment of migrating interneurons

A-C. Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos (A), in the CM from E14 cortical cells in culture for 4 days (B), in the CM from hCOs after 4 days in culture (C). The CM was collected from hCOs at different time points in culture (d19, d38, d60, d80). Protein extract (PE) from 60 days hCOs was used as a positive control. GAPDH immunostaining was used as a negative control on hCOs CM. Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM samples

D. Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-empty vector or -Cstb, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.

E. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex-Bin1 corresponded to apical side and Bin5 to pial side. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles.

F. Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry and HA-Cstb expressing vector, analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.

G. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro-caudally.

Data information: Scale bars: 100 µm (D and F), 20 µm in (d' and f'). Data are represented as mean ± SEM. Statistical significance was based on Mann-Whitney test (*P < 0.05). Every dot in the plots refers to independent analyzed mouse brains

Exact p-values in Appendix Table S1.
CystatinB is essential for proliferation and interneuron migration in individuals with EPM1 epilepsy (2020)
Figure 5

Figure 5. Proliferation is reduced in EPM1-derived cerebral organoids in a cell non-autonomous manner

A. Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points. Right panel shows the difference of the areas of f-CTRL, b-CTRL, UL1 and UL4 hCOs at different time in culture. Data shown as Z-scores relative to the mean of b-CTRL.

B. Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles.

C,D. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricles like structures and of the number of PH3+ cells facing the ventricle/apical surface length (µm). Data shown as Z-scores relative to the mean of b-CTRL.

E. Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles.

F,G. Quantification of the total number of proliferating KI67+ cells/ area of ventricles like structures and of the number of PH3+ cells facing the ventricle/the apical surface length (µm). Data shown as Z-scores relative to the mean of CTRL.

H. Scheme of the experiment with conditioned medium exchange between b-CTRL and UL4 hCOs and quantification of the Ki67+ cells by FACS analysis on 36 days old dissociated hCOs. Four samples were analyzed as biological replicates. FACS plots in Fig.EV4G.

Data information: Nuclei (blue) are stained with DAPI. Scale bars: 200 μm in (A); 50 μm in (B and E). Data are represented as mean ± SEM. Statistical significance was based on Mann-Whitney test (*P < 0.05, ***P <0.001). Every dot in the plots refers to independent analyzed ventricles per hCO from at least 3 different hCOs generated in at least 2 independent batches.

Exact p-values in Appendix Table S1.
CystatinB is essential for proliferation and interneuron migration in individuals with EPM1 epilepsy (2020)
Figure 6

Figure 6. EPM1-derived cerebral organoids exhibit premature differentiation. Proteomic analysis in patients' hCOs

A. Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for the neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles.

B. Quantification of the DCX+ area. The area of DCX+ cells was calculated and related to the total area (µm2) of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL.

C. Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles.

D. Quantification of the DCX+ area. The area (µm2) of DCX+ cells was calculated and related to the total area of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL.

E. Quantification of the number of neuronal DCX+ cells by FACS sorting on 60 days old dissociated hCOs. Data shown as Z-scores relative to the mean of b-CTRL. FACS plots in Fig.EV5A.

F. Micrograph sections of d30 fused-organoid; ventral GFP-f-CTRL and dorsal UL1.

G. Micrographs of sections from d30 fused organoids, showing ventral GFP+ cells in the dorsal area. The migrating GFP+ cells were counted starting from 100 µm far from the ventral/dorsal border.

H. Quantification of ventrally-derived GFP+ cells in dorsal/thickness of the counted area (µm3). The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig.EV5D. Every dot represents a different hCOs and different areas were counted for each hCO.

I. Volcano plot representing proteins identified by high-resolution nanoLC MS/MS analysis. Up- and down-regulated proteins (-0.3≥log2FC≥0.3) in human cerebral organoids (UL1 vs b-CTRL) are shown in red and green, respectively.

J. GOChord plot showing relationships between selected representative biological process GO terms and differentially expressed proteins identified by nanoLC MS/MS analysis. The representation shows up-(red) and down-regulated (green) proteins differentially expressed in UL1 relative to b-CTRL (-0.3≥log2FC≥0.3).

Data information: Nuclei (blue) are stained with DAPI. Scale bars: 50 µm in (A and C); 200 µm in (F) and 100 µm in (G). Data are represented as mean ± SEM. Statistical significance was based on Mann-Whitney test (*P < 0.05, ***P <0.001). Every dot in the plots refers to independent analyzed ventricles per hCO from at least 3 different hCOs generated in at least 2 independent batches.

Exact p-values in Appendix Table S1.
SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity (2021)
Figure 2

Figure 2. HCR-ICC detects co-localization of SRSF1 and CGG repeat RNA.

(A) Hybridization Chain Reaction (HCR) detection of CGG repeat RNA along with immunocytochemistry (ICC) to detect SRSF1-FLAG in U2OS cells. Expanded view of merged channels shows the CGG RNA foci (arrowhead) and an example of SRSF1-CGG RNA foci co-localization (arrow). DAPI marks the nucleus. Scale bars are 10 µm.

(B) Co-localization analysis of CGG RNA (red traces) and SRSF1 (green traces) showing overlaps of CGG RNA and SRSF1. (a.u. = arbitrary unit)

(C) Co-immunoprecipitation of indicated SRSF proteins with PP7-tagged control sequence (partial nLUC sequence) to correct for non-specific binding, AUG and CGG reporter RNAs. SRSF1 and 2 specifically immunoprecipitated with PP7-tagged CGG reporter.
SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity (2021)
Figure 3

Figure 3. SRSF proteins act as modifiers of CGG and GGGGCC-repeat associated toxicity in Drosophila.

(A) Schematic of (CGG)90-EGFP construct and experimental outline for rough eye phenotype screening.

(B) Quantitation of GMR-GAL4 driven uas-(CGG)90-EGFP eye phenotype with candidate modifiers (n > 30 flies/ genotype). siNTC = siRNA against a non-targeting control gene (mCherry). Different siRNA lines for the same target gene are numbered (#1 and #2). Error bars represent mean +/- SD.

(C) Representative photographs of fly eyes expressing either GMR-GAL4 driver alone or with uas-(CGG)90-EGFP construct, with fly SRSF1 (dSF2) and SRSF2 (dSC35) knockdown or disruptions (insertion).

(D) Representative photographs of fly eyes and quantitation (below) of rough eye scores with fly SRSF1 overexpression (dSF2 OE); n=20-32/genotype. Error bars represent mean +/- SD.

(E) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with indicated uas-siRNAs against fly SRSF genes in comparison with non-targeting control (NTC) siRNA against LUC/luciferase.

(F) Quantitation of (GGGGCC)28-EGFP rough eye phenotype with SRSF modifiers (n > 30 flies/ genotype). Error bars represent mean +/- SD.

(G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP at 29˚C along with the quantifications of necrosis and eye width. n=28-30/genotype. Error bars represent mean +/- SD.

(H-I) Survival assays of flies expressing (CGG)90-EGFP under Tub5-GS (H) and ELAV-GS (I) drivers with control or SRSF1 siRNAs. Expression of (CGG)90-EGFP was initiated with addition of drug starting 1 day post eclosion and continued through experiment (Log-rank Mantel-Cox test; n=98-101/genotype for Tub-GS and n=120-141/genotype for ELAV-GS flies) ∗p < 0.05, ∗∗p < 0.01.

(J) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel-Cox test; n=71-93/genotype) with control or SRSF1 siRNAs. ∗∗p < 0.01.

Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling. Details of fly genes are described in Appendix Table S2.
SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity (2021)
Figure 4

Figure 4. SRPK1 knockdown modifies CGG and GGGGCC repeat-associated toxicity in Drosophila.

(A, B) Representative (A) photographs of fly eyes and (B) quantitation with siRNA mediated knockdown of SRPK1 (dSRPK1); n=30-34/genotype. Error bars represent mean +/- SD.

(C) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion.

(D) Quantitation of rough eye phenotypes. t-test with Welch corrections for comparisons with the control; n= 31-34 flies/ genotype. Error bars represent mean +/- SD.

(E) Representative external eye imaging for detection of GFP aggregates caused by (CGG)90-EGFP transgene expression (top). Converted images used to quantify total intensity of GFP puncta (bottom).

(F) Depletion of SRSF1 or SRPK1 by RNAi results in reduced (CGG)90-EGFP puncta compared to control siRNA as quantified by total intensity (a.u. = arbitrary unit). n=13-15 flies/genotype. Error bars represent mean +/- SD.

Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.