EMBO/soda-nel huggingface dataset:
LIF is a mitogen for choroidal EC and protects  choriocapillaris from  injury: implication for  AMD (2021)
Figure 5

Figure 5. Effects of LIF and other IL-6 family members inin mouse ocular models.

A). LIF enhanced laser-induced CNV. Intravitreal administration of LIF (10 ng and 100 ng, Sigma) and OSM (10 ng) after laser-induced CNV. PBS was used as vehicle control. OCT-A imaging and immunostaining of choroid flatmounts was carried out 10 days after laser induction. Quantification of CNV area is shown. Dashed line circles indicate the CNV in OCT-A images. n = 5. Dot plot shows all data points from three independent experiments. Scale bar = 100 µm.

B) Sodium Iodate was administered to induce retinal and CC damage in mice. After sodium iodate injection, the indicated amounts of LIF, CT-1 or OSM were injected in the eyes. Choroid capillaries were imaged by OCT-A system, n=5. The avascular area in CC is indicated in yellow.

C.) H & E staining of eyes treated with different IL-6 proteins following sodium iodate injection (n = 5). Representative images are shown. Scale bar = 100 µm.

D). Avascular area in choroid in sodium iodate model was determined and quantified using Image J.

E). Thickness of retina in H&E staining was quantified and bar graph was shown using ImageJ.

Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.
LIF is a mitogen for choroidal EC and protects  choriocapillaris from  injury: implication for  AMD (2021)
Figure 6

Figure 6. Expression of LIF and LIFR in ECs.

(A) Q-PCR (Taqman) analysis of LIF expression in BCE cells and bovine pericytes (bPericyte).

(B) Bovine LIF ELISA of BCE cells and bovine pericyte. Cells were cultured for 48 hrs before harvesting the medium.

(C) LIFR expression in BCE cells and bovine pericyte by qPCR (Taqman).

(D) Image of FACS selection of CD13+/CD45- and CD31+/CD45- cells.

(E) Immunostaining for CD31, CD13 and DAPI inin isolated primary mouse cells before and after FACS selection.

(F) Mouse LIF expression in FACS-selected cells.

(G) Human choroidal EC (HCEC) and mouse retinal EC (MREC) were treated with LIF 10 ng, LIF 100 ng for 30 min. (PBS was used as control). STAT3 phosphorylation was detected by western blot.

(H) H&E staining of LIFR and CD31 in mouse eye sections. The magnified images of the dashed line boxes, a and b, are shown on the right panel. Box a shows LIFR staining in retina and box b showed LIFR staining in RPE/choroid layer.

(I) Whole mount staining of LIFR and CD31 in mouse retina. The fluorescent staining for CD31 (green) and LIFR (red) is shown. White arrows indicate the co-staining of CD31 and LIFR on retinal ECs.

Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOV was used as statistical test.
LIF is a mitogen for choroidal EC and protects  choriocapillaris from  injury: implication for  AMD (2021)
Figure 7

Figure 7. LIFR, but not LIF, is highly expressed in human choroidal ECs by single-cell transcriptomic analysis.

(A-E) Violin plots of the log scale normalized expression of LIF receptor subunits, IL6ST (A), LIFR (B), LIF (C), IL6R (D) and KDR (E) in all cell types in the human choroid (based on (Voigt et al., 2019)). Note the wide distribution of IL6ST (gp130), the common subunit to all receptors of the IL-6 family (A). In contrast, LIFR is strongly expressed in ECs and to a lesser extent in fibroblasts and pericytes (B). LIF is expressed in fibroblasts, macrophages/monocytes and pericytes and minimally in ECs (C). The specific IL-6 receptor (IL6R) is highly expressed in macrophage/monocytes (D), while LIFR has little expression in these cells (B). KDR shows, as expected, high expression selectively in EC (E).

(F) Cell clusters in uniform manifold approximation and projection (UMAP) space.
Efficient plasmid transfer via natural competence in a microbial co-culture (2023)
Figure 2

Figure 2. The presence of recA in the E. coli donor enhances HGT efficiency in the co-culture.

(A) Bar plot of HGT efficiencies of the pBB275 plasmid in the co-culture composed of a E. coli recA+ or recA- donor and engineered B. subtilis at 6 hr.

(B, C) (B) Extracellular pBB275 plasmid concentration or (C) extracellular E. coli genomic DNA concentration at 0 or 6 hr in the co-cultures composed of a recA+ or recA- E. coli plasmid donor and engineered B. subtilis.

(D) Histogram of GFP expression driven by a SOS response promoter in E. coli MG1655 ∆recA, E. coli MG1655, E. coli MG1655 harboring pUC19, E. coli MG1655 harboring pBB275 or E. coli MG1655 ∆sulA RecA E38K in monoculture measured by flow cytometry. Inset: bar plot of the fraction of E. coli expressing high GFP (>10000 a.u.) for each strain.

(E) Extracellular pBB275 or pUC19 plasmid concentration in the E. coli MG1655 monoculture or or co-culture with engineered B. subtilis.

(F) HGT efficiencies of pBB275 plasmid (with or without a f1 ori) at 6 hr in the co-culture composed of E. coli MG1655 and engineered B. subtilis.

(G) Schematic of proposed model for the efficient plasmid transfer via natural competence in the co-culture composed of a recA+ E. coli donor and recipient B. subtilis. The plasmid leads to the activation of the SOS response, which in turn enhances eDNA release and plasmid multimerication via RecA. B. subtilis enhances E. coli eDNA release even being inhibited by E. coli.

(H) Relationship between total homology length and transformation frequency of pBB275 plasmid in the co-culture composed of E. coli MG1655-rfp and engineered B. subtilis or B. subtilis monoculture at 6 hr. In the B. subtilis monoculture, 1 µg/mL pBB275 plasmid DNA derived from E. coli DH5α was introduced.

(I) Relationship between total homology length and HGT efficiency of the pBB275 plasmid in the co-culture composed of the DAP-auxotrophic E. coli BW29427 and wild-type B. subtilis PY79, 168 or natto IFO3335 at 6 hr. HGT efficiency of pBB275 plasmid with total ~200 bp and ~1000 bp homology for B. subtilis natto was below the detection limit.

Data information: All experiments had three biological replicates. Bars and lines are the average of the biological replicates.
Balancing the length of the distal tip by Septins is key for stability and signalling function of primary cilia (2021)
Figure 1

Figure 1. Characterisation of proxmal/distal cilia segmentation in RPE1 cells.

a-b, Representative images of cilia (a) and dot plots (b) showing primary cilia length variation at different time points after serum starvation (SS). Markers in (a) show ARL13B (cilia) and PCNT (Basal body). The graph shows individual length of cilia from 3 biological replicates (n>150 cilia per sample and experiment). Scale bar: 1µm.

c, Percentage of ciliated RPE1 cells after SS from a. 3 biological replicates, n>100 cells per sample and experiment repetition.

d, Representative images show cilia segmentation based on acetylated tubulin (Ac tub), polyglutamylated tubulin (Glu tub), ARL13B and PCNT. The cartoon on the left indicates proximal segments (PS, Glu tub rich-region) and distal segments (DS). DS were determined by calculating the difference between PS and total cilia length based on ARL13B staining as depicted by the dashed lines on the images. Scale bar: 1.5µm.

e, Quantification of d. Comparative analysis of average cilia length based on Ac tubulin and ARL13B (cilia) staining shown for specified cilia length groups (2<, 2-3, 3-4 and >4µm). Three biological replicates, n=400 cilia per sample and repetition. The numbers above the cartoons show the ratio of the calculated Ac tub/whole cilia length.

f, Average length of PS and DS for the cilia length groups depicted. Representative images (d) and quantifications of PS (Glu tub) and whole cilia (ARL13B) length are shown. The numbers above the cartoons show the ratio of the calculated DS/whole cilia length. Three biological replicates, n>650 cilia per sample and repetition.

g, Percentage of cilia with depicted DS length after 8h and 48h SS. Three biological replicates, n>150 cilia per sample and repetition.

Data shown in (b, c, e, f and g) include mean ±s.d. and P values of (g) are calculated by two-tailed unpaired student t-test. See source data.
Balancing the length of the distal tip by Septins is key for stability and signalling function of primary cilia (2021)
Figure 2

Figure 2. Microtubule perturbators and SEPT2 depletion change only the distal segment length in RPE1 cells.

a, Representative images of 48h serum starved RPE1 cells treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before immunostaining with the indicated antibodies. Scale bar: 3µm.

b, Quantification of (a) showing average length of proximal segment (PS, Glu tub) and whole cilia (ARL13B). The numbers above the cartoons show the ratio of the calculated distal segment (DS)/whole cilia length. Three biological replicates, n=100 cilia per sample and repetition.

c-d, RPE1 cells were treated with the indicated siRNAs before serum starvation for 48h. Representative immunostained images with indicated antibodies (c) and quantifications of PS (Glu tub) and whole cilia (ARL13B) length (d) are shown. The numbers above the cartoons in (d) show the ratio of the calculated DS/whole cilia length. Scale bar: 2µm. Three biological replicates, n=100 cilia per sample and repetition.

e-f, Cells were treated with the indicated siRNAs before serum starvation for 48h and treatment with DMSO or Noco for 3h before immunostaining with the indicated antibodies. Representative images (e) and quantification of cilia length based on ARL13B (f) are shown. Scale bar: 2µm. Three biological replicates, n=100 cilia per sample and repetition.

Data shown in (b, d and f) include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test. See source data.
Balancing the length of the distal tip by Septins is key for stability and signalling function of primary cilia (2021)
Figure 3

Figure 3. Endogenous Septin localises inside the cilium and upper part of the TZ in a microtubule-dependent manner.

a, Images show the localisation of endogeneous SEPT2 and SEPT7 in RPE1 ARLB13B-mRuby2 cells with basal body staining. The insets to the right show enlargements of the cilium. Scale bar: 4µm (large) and 2µm (small).

b, STED microscopy images showing ciliated RPE1 cells expressing EGFP-SEPT2 or SEPT7-EGFP and stained for ARL13B. Scale bar: 1µm.

c, Relative signal intensities were determined along the cilium for the indicated proteins at the ciliary tip (T), middle (M) and base (B) regions as specified in (b) (white lines).

d, Representative images showing co-localisation of MKS3 or CEP290 in ciliated RPE1 ARLB13B-mRuby2 and GFP-SEPT2 cells. The white arrows point to the specific MKS3 or CEP290 signals. Scale bar: 1µm.

e, Line scan measurements show the normalised signal intensities along the cilium for the indicated proteins as specified in (d) (dashed lines).

f, Effect of nocodazole (Noco) treatment (3h) on the localisation of endogenous SEPT2 and SEPT7 at cilia in RPE1 cells. The insets show magnifications of the cilium. Scale bar: 5µm (large) and 2.5µm (small).

g, Relative signal intensity of endogenous SEPT2 or SEPT7 along the cilium from (f). SEPT2 or SEPT7 signals at cilia were normalized to the total cilia length. Three biological replicates, n>90 cilia per sample and repetition.

Data show in (g) includes mean ±s.d.; P values are calculated by two-tailed unpaired student t-test. See source data.
Balancing the length of the distal tip by Septins is key for stability and signalling function of primary cilia (2021)
Figure 4

Figure 4. Septin negatively controls distal segment elongation in RPE1 cells.

a-b, Cilia length changes in RPE1 cells treated with the indicated siRNAs and 48h SS stained with indicated antibodies. Representative images of cilia (a) and quantifications (b) are shown. Dot plots in (b) represent individual cilia. Scale bar: 1.5µm. Three biological replicates, n=100 cilia per sample and repetition.

c-d, Cilia length changes in RPE1 SEPT2-KO single clones after 48h SS stained with indicated antibodies. Representative images of cilia (c) and quantifications (d) are shown. Dot plots in (d) represent individual cilia. The parental cells (WT) and four KO clones (C7, C16, C10, C13) derived from two independent gRNAs (g1 and g2, as depicted in Appendix fig. 3e) were analysed. Three biological replicates, n>80 cilia per sample and repetition. Scale bar: 2µm.

e-f, Cilia segmentation in SEPT2-KO ciliated cells serum starved for 48h. Cells were immunostained with the indicated antibodies. Representative images (e) and quantifications of proximal segment (PS, Glu tub) and whole cilia (ARL13B) length (f) are shown. The numbers above the cartoons in (f) show the ratio of the calculated distal segment (DS)/whole cilia length. Scale bar: 2µm. Three biological replicates, n=100 cilia per sample and repetition.

g, Electron microscopy of RPE1 WT and SEPT2-KO cilia. Black arrows point to the axoneme and basal body (BB). Scale bar: 400nm.

h, Quantification of (g). Only cilia detected from bottom (BB) to tip in serial sections were quantified. N>15 cilia from two independent experiments.

i-j, RPE1 WT and SEPT2-KO cells after 48h SS were treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before immunostaining with the indicated antibodies. Representative images (i) and quantifications of PS (Glu tub) and whole cilia (ARL13B) length (j) are shown. The numbers above the cartoons in (j) show the ratio of the calculated DS/whole cilia length. Scale bar: 3µm. Three biological replicates, n=100 cilia per sample and repetition.

Data show in (b, d, f, h, j) include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test (in f, j) or by unpaired Wilcoxon-Mann-Whitney Rank Sum Test (in b, d, h). See source data.
Balancing the length of the distal tip by Septins is key for stability and signalling function of primary cilia (2021)
Figure 5

Figure 5. Septin is required for the KIF7 accumulation at the cilia tip to suppress distal segment growth.

a-b, Representative images show the localisation of IFT88, KIF17 and KIF7 in RPE1 WT and SEPT2-KO cells after 48h serum starvation (SS) stained with indicated antibodies (a) and quantification of the relative signal intensities for the indicated proteins (b). White arrows point to the base and tip localisation of KIF7. Scale bar: 3µm. IFT88 and KIF17 signal intensities were normalized to the whole cilia length. KIF7 signal intensity was quantified at ciliary tip or base as indicated in (a) (white arrows). Three biological replicates, n>80 cilia per sample and repetition.

c-d, KIF7 localisation at the tip and base of cilia (indicated by arrows) in RPE1 cells after 48h SS treated for 3h with solvent control (DMSO) or nocodazole (Noco) and quantification of the relative signal intensities for the ciliary base and tip of KIF7 (d). Scale bar: 2µm. KIF7 signal intensity was quantified at the ciliary tip or base as indicated in (c) (white arrows). Three biological replicates, at least 80 cilia per sample and repetition.

e-f, RPE1 WT and SEPT2-KO cells were treated with control or KIF7 siRNA after 48h SS and immunostained with the indicated antibodies. Representative images (e) and quantifications of proximal segment (Glu tub) and whole cilia (ARL13B) length (f) are shown. The numbers above the cartoons in (f) show the ratio of the calculated distal segments /whole cilia length. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm.

Data shown in (b, d, f) include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test. See source data.
Balancing the length of the distal tip by Septins is key for stability and signalling function of primary cilia (2021)
Figure 6

Figure 6. Functional transition zone (TZ) allows to transfer KIF7 to the ciliary tip in RPE1 cells.

a, Localisation of MKS3, CEP290 and NPHP1 in RPE1 WT and SEPT2-KO after 48h serum starvation (SS) are immunostained with CEP164 (distal appendages), ODF2 (sub-distal appendages), and ARL13B (cilia). White arrows point to the specific signals for MKS3 or CEP290. The white star points to the unspecific MKS3 signal around the centrosome. Low and high exposure times for MKS3m CEP290 and NPHP1 are shown as indicated in the upper panel. Scale bar: 1µm.

b-c, Quantification of (a). The length obtained for MKS3 along the cilia (b) and the relative signal intensity of the indicated proteins at the ciliary base (c) are shown. Three biological replicates, n=100 cilia per sample and repetition.

d, FRAP experiment of SSTR3-GFP in RPE1 WT and SEPT2 KO cells. Cells were starved for 48h before the beginning of inspection and imaged every 30sec for 10mins after bleaching. Scale bar: 5µm. For videos, see Movie EV1 and 2.

e, Percentage of normalised SSTR3-GFP signal recovery at whole cilia after photobleching from (d). n=20 cilia are quantified from three biological replicates

f-g, Influence of MKS3 depletion on cilia length of RPE WT and SEPT2-KO cells. Cells are treated with the indicated siRNA and serum starved (SS) for 48h before immunostaining with the indicated antibodies. Representative images (f) and quantifications of proximal segment (Glu tub) and whole cilia (ARL13B) length (g) are shown. The numbers above the cartoons in (g) show the ratio of the calculated distal segments/whole cilia length. Scale bar: 2µm. Three 3 biological replicates, n=100 cilia per sample and repetition.

h-i, A functional TZ is required for KIF7 cilia tip localisation in RPE1 cells. KIF7 signal intensities are measured in ciliated RPE1 cells treated with control or MKS3 siRNA and 48h SS. Representative images (h) and quantification of KIF7 signal intensity at the base and tip of cilia (h) are shown. Scale bar: 2µm. Three biological replicates, at least 60 cilia per sample and repetition.

Data shown in (b, c, e, g, i) include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test. See source data.
Balancing the length of the distal tip by Septins is key for stability and signalling function of primary cilia (2021)
Figure 7

Figure 7. Over-extended distal segments are more prone to undergo ectocytosis.

a, Time-lapse images of RPE1 WT and SEPT2-KO cells stably expressing ARL13B-GFP and γ-tubulin-mRuby2. Cells are starved for 32h before the beginning of inspection and imaged every 15min for 9.5h. Scale bar: 5µm. For videos, see Movie EV3 and 4.

b, Quantification of (a) showing changes in cilia length during time-lapse imaging. Three biological replicates, 10 cilia per sample and repetition.

c, Time-lapse images show ectocytosis events observed in RPE1 SEPT2-KO cells. Experiments are done as in (a) with exception that cells are imaged every 15min. Scale bar: 2.5µm. For videos, see Movie EV9 and 10.

d, Percentage of cilia that remained intact, underwent ectocytosis during live-cell imaging from (a) and (c). Three 3 biological replicates, n>100 cilia per sample and repetition.

e-f, Determination of the cilia breakage point during live-cell imaging of RPE1 SEPT2-KO ciliated cells. The ectocytosis point was calculated by dividing the cilia length at the time of breakage (b, t0) by the length of the cilium one time-point prior to t0 (a, t-1), as depicted in (e). The graph in (f) shows that breaking points (b/a) higher than 0.5 (i.e. breakage towards the cilia tip) are more frequent. Three biological replicates, n=50 cilia per sample and repetition.

g-h, RPE1 SEPT2-KO cells expressing ARL13B-mRuby2 and Neongreen-EFHC1 after 48h SS and immunostained with Glu-tub antibody. Representative images (g) and quantifications of proximal segment (Glu tub), EFHC1 and whole cilia (ARL13B) length (h) are shown. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm.

i, Time-lapse images of RPE1 SEPT2-KO cells stably expressing ARL13B-mRuby2 and Neongreen-EFHC1. Cells were starved for 32h before the beginning of inspection and imaged every 15min for 9.5h. The point of ectocytosis and the the fragmented cilia are indicated by the white arrow and star, respectively. Scale bar: 5µm. For videos, see Movie EV13.

j, Quantification of (i) showing that (f)-ectocytosis mainly occurs at EFHC1-poor region of cilia during time-lapse imaging. Three biological replicates, 50 cilia per sample and repetition.

Data shown in (d, h, j) include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test. See source data.
SHLD2 promotes class switch recombination by preventing 
inactivating deletions within the Ig locus (2020)
Figure 1

Figure 1. SHLD2 does not affect lymphocyte development or V(D)J recombination.

A. Schematic of the Shld2 exon 4 showing the 3 sgRNAs used to produce a 251 base pair deletion in one founder line that leads to a frameshift and usage of a premature stop codon.

B. Characterization of the various B cell progenitor fractions as described by [38] in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls. Gating strategy for measuring these populations is shown in Figure EV1A.

C. Characterization of the indicated thymic T cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls. Gating strategy for measuring these populations is shown in Figure EV1D.

D. Characterization of the indicated immature and mature splenic B cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls. Gating strategy for measuring these populations is shown in Figure EV1B.

E. Characterization of the indicated peritoneal B cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls. Gating strategy for measuring these populations is shown in Figure EV1C.

F. Left panel: Schematic of the A70.2 INV-4 cell line strategy to induce RAG1/2-mediated recombination using imatinib. Right panel: A70.2 INV-4 cell lines were transduced with lentiviruses encoding the lentiCRISPRv2 expressing sgRNAs against the 53bp1, Shld1, Shld2, Shld3, and Lig4 genes. Guide RNA targeting chicken AID was used as a negative control (Ctrl). Cells were selected with puromycin and treated with 3 μM imatinib for 4 days after which GFP frequency was measured (mean ± SD of 3 biological replicates). The insertion-deletion (indel) penetrance as measured by TIDE analysis [39] of sequence for each of these sgRNA constructs is shown in Figure EV1E, and the baseline GFP frequency prior to imatinib stimulation is shown in Figure EV1F. sgRNA sequences used are shown in Table EV2.
SHLD2 promotes class switch recombination by preventing 
inactivating deletions within the Ig locus (2020)
Figure 2

Figure 2. SHLD2-deficient mice have defects in class switch recombination.

A. Concentration of the various indicated isotypes in the serum of 6-8 week old unimmunized WT and Shld2−/− mice. Values are mean concentration ± SD of 4 biological replicates; ** P ≤ 0.01, unpaired two-tailed t-test.

B. B cells were purified from spleens from WT and Shld2−/− mice, and stimulated to undergo CSR to the various indicated isotypes using different stimulation cocktails [33]. Cells were then analyzed by flow cytometry for expression of the various indicated isotypes and the percent expression of each isotype are reported. Values are mean frequency ± SD of 4 biological replicates; **** P ≤ 0.0001, unpaired two-tailed t-test.

C. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG and the serum was withdrawn 2 weeks post immunization and serial dilutions were subjected to ELISA analysis for NP-specific antibodies of the indicated isotypes. Values are mean absorbance ± SD of 4 biological replicates.

D. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG, spleens were isolated and anti-IgG secreting cells were enumerated by the ELISPOT assay. Values are mean frequency ± SD of 4 biological replicates, except for Shld2-/- (n=3); * P ≤ 0.05, ** P ≤ 0.01, unpaired two-tailed t test.