EMBO/soda-nel huggingface dataset:
CK1-mediated phosphorylation of FAM110A promotes its interaction with mitotic spindle and controls chromosomal alignment (2021)
Figure 2

Figure 2. Expression of FAM110A increases towards mitosis

RPE cells were transfected with control or FAM110A siRNA, whole cell lysates were prepared after 48 h and indicated antibodies were tested in immunoblotting.

RPE cells were released for indicated times from a thymidine block and collected cells were analyzed by immunoblotting and FACS. Nocodazole was added after 4 h to arrest cells in mitosis. Alternatively, RO3306 inhibitor was added to prevent mitotic entry. Where indicated sample was collected by mitotic shake-off (MS) to obtain pure mitotic population. Samples were analyzed by immunoblotting (upper panel). Staining for pS10-H3 was used as a marker of mitosis, TFIIH as a loading control. In parallel, cell cycle progression was followed by flow cytometry using DAPI and mpm2 staining. Representative plot is shown, M indicates percentage of mitotic cells (middle panel). Signal of the rabbit FAM110A antibody was quantified in three independent replicates and normalized to the loading and to the thymidine arrested sample (lower panel). Bars show median ± SD.

RPE cells were arrested in mitosis by nocodazole, collected by mitotic shake-off and released to fresh media for indicated times to allow mitotic exit. Loss of pS10-H3 signal was used as marker of mitotic exit.

RPE cells arrested in G2 by RO3306 (G2) or in mitosis (M) by nocodazole treatment were extracted and endogenous FAM110A was immunoprecipitated using rabbit polyclonal antibody immobilized on pA/G beads. Beads were incubated with mock or with lambda phosphatase for 20 min at 30°C. Proteins were separated by SDS-PAGE and detected by immunoblotting.

RPE cells transfected with control or FAM110A siRNA were fixed with 4 % PFA, probed with rabbit polyclonal antibody against FAM110A and mouse monoclonal to γ-tubulin, and were analyzed by confocal microscopy. Shown is maximal projection of representative mitotic (upper panel) and interphase (lower panel) cells. Scale bars indicate 10 μm.

Quantification of the signal at spindle poles from (E). Signal intensity of the rabbit polyclonal antibody to FAM110A was determined in regions positive for γ-tubulin. Each dot represents a single spindle pole. Bars show median ±SD. Significance was evaluated by a t-test (n=3), ****p<0.0001.

Asynchronously growing RPE cells were fixed and probed with the rabbit polyclonal antibody to FAM110A. Shown are representative images of cells in various phases of mitosis. Individual z-stacks were de-convoluted using Huygens Deconvolution Software, processed sequentially and are presented as maximum projections. Scale bars indicate 10 μm.
CK1-mediated phosphorylation of FAM110A promotes its interaction with mitotic spindle and controls chromosomal alignment (2021)
Figure 3

Figure 3. Depletion of FAM110A impairs progression through mitosis

RPE cells transfected with control or FAM110A siRNA were fixed, stained with MPM2 antibody (marker of mitosis) and DAPI, and were analyzed by flow cytometry. Representative graphs are shown. Numbers indicate fractions of individual cell cycle phases and mitotic cells.

Quantification of the mitotic index in three experiments from (A) and in RPE cells transfected with siRNAs to FAM110B and CSPP1 that were analyzed in parallel. Shown is median ±SD (n=3). Statistical significance was determined by ANOVA (* p<0.05, ** p<0.005).

RPE cells transfected with control, FAM110A or CSPP1 siRNAs were collected and analyzed by immunoblotting with indicated antibodies.

RPE cells stably expressing mouse EGFP-FAM110A were transfected with control or two various FAM110A siRNAs and whole cell lysates were analyzed by immunoblotting with indicated antibodies (left panel). Empty arrowhead indicates position of the endogenous FAM110A, filled arrowhead the EGFP-FAM110A. In parallel, mitotic index was determined by flow cytometry. Statistical significance was evaluated by ANOVA (n=3). Bars indicate median ± SD.

RPE cells transfected with control or FAM110A siRNA were grown for 48 h and treated with MG132 30 min prior fixation to trap mitotic cells in metaphase. Impaired chromosomal alignment and multipolar spindles were scored and representative images are shown. Scale bars indicate 20 μm, arrowheads point to misaligned chromosomes. More than 30 metaphases were evaluated per condition (n=3). Bars indicate median ±SD. Statistical significance was determined by two-tailed t-test, (*** p<0.001, * p<0.05).

Quantification of lagging chromosomes in anaphase cells from (E). More than 30 anaphases were evaluated per condition (n=3). Representative images of abnormal anaphase are shown, scale bars indicate 20 μm, arrowheads point to incorrectly segregating chromosomes. Statistical significance was determined by two-tailed t-test. Bars indicate median ±SD (*** p<0.001).
CK1-mediated phosphorylation of FAM110A promotes its interaction with mitotic spindle and controls chromosomal alignment (2021)
Figure 4

Figure 4. Depletion of FAM110A delays progression through mitosis

U2OS cells stably expressing H2B-EGFP were transfected with indicated siRNAs and after 48 h they were filmed in 5 min intervals. Time frame closest to the nuclear envelope breakdown (NEB) was set as 0 min. Representative examples are shown (n=3). Black bottom line indicates the time frames from NEB to the metaphase-to-anaphase transition. Scale bars indicate 10 μm.

Progression through mitosis was categorized in cells from (A) (100/condition) as follows: nuclear envelope breakdown to the metaphase-to-anaphase transition (MAT), anaphase, telophase and interphase. Data from one of three experiments are shown, each bar indicates one cell. Cells that died during imaging are shown in grey.

Time from the nuclear envelope breakdown to MAT was quantified in cells from (B). Shown is median ±SD (n=3). Statistical significance was determined by ANOVA (****p<0.0001).

Abnormal nuclear organization was scored in cells from (B) that completed cell division. Representative images of aberrant nuclei are shown. Scale bar indicates 15 μm. Plotted is percentage of cells with aberrant nuclei. Shown is median ± SD (n=3). Statistical significance was determined by ANOVA (*** p<0.001).
CK1-mediated phosphorylation of FAM110A promotes its interaction with mitotic spindle and controls chromosomal alignment (2021)
Figure 5

Figure 5. FAM110A interacts with CK1, tubulin and actin during mitosis

Cell extracts from RPE cells stably expressing EGFP or EGFP-FAM110A arrested in mitosis by nocodazole were incubated with GFP Trap. Bound proteins were analyzed by mass spectrometry. Shown are top ten proteins that that were significantly enriched in EGFP-FAM110A complex compared to EGFP in three independent experiments. Proteins TUBB8 and TUB1AC were also significantly enriched in complex with EGFP-FAM110A. Number indicates the fold change interaction compared to the control.

Cell extracts from asynchronously growing or mitotic RPE-EGFP or RPE-EGFP-FAM110A cells were incubated with GFP Trap and bound proteins were analyzed by immunoblotting. Antibody against pS10H3 was used as a marker of mitosis.

Cell extracts from asynchronously growing or mitotic RPE-EGFP-FAM110A cells were incubated with CSNK1D antibody or control IgG immobilized on protein A/G beads. Bound proteins were probed with antibody against GFP or CK1δ. Staining for pS10-H3 was used as marker of the mitotic population.

Scheme of the EGFP-FAM110A constructs used in the study. Numbering is based on human FAM110A. N, C, and Pro represent N-terminal, C-terminal and Pro rich domains, respectively.

Cells were transfected with indicated constructs and after 48 h they were lysed in RIPA buffer. Cell extracts were immunoprecipitated with GFP Trap and probed by immunoblotting (n=2).

Cells were transfected with EGFP, EGFP-FAM110A-WT or EGFP-FAM110A-FA. Cell extracts were immunoprecipitated with GFP Trap and binding of CK1 was determined by immunoblotting (n=3).
CK1-mediated phosphorylation of FAM110A promotes its interaction with mitotic spindle and controls chromosomal alignment (2021)
Figure 6

Figure 6. CK1 phosphorylates the C-terminal domain of FAM110A during mitosis

RPE cells arrested in mitosis by nocodazole (NZ) were treated with DMSO, PLK1 inhibitor BI2536 (100 nM), Aurora-A inhibitor MLN8054 (0.25 μM), CDK1 inhibitor RO3306 (20 nM) or CK1 inhibitor PF-670462 (1 μM) for 60 min, collected and analyzed by immunoblotting with indicated antibodies. Asynchronously growing RPE cells are shown for comparison (left line). Staining for H3-pS10 served as a marker of mitosis (n=3).

Isolated EGFP-FAM110A-WT or -∆C proteins were incubated with active CDK1/GST-cyclin B1 in a kinase buffer containing 32P-γATP for 20 min at 30°C. Phosphorylation was detected by autoradiography and position of FAM110A by immunoblotting. Empty arrowhead indicates position of CDK1.

RPE cells were transfected with control, CSNK1D or CSNK1E siRNA and nocodazole was added 12 h before collecting the cells by mitotic shake off. Whole cell lysates were probed with indicated antibodies.

EGFP-FAM110A-WT, -∆Pro or -∆C proteins isolated from transfected HEK293 cells using GFP-trap were incubated with mock or with active GST-CK1δ for 20 min at 30°C. Mobility of the proteins was analyzed by immunoblotting (n=2).

Cells expressing the wild-type EGFP-FAM110A or its mutants were arrested in mitosis by nocodazole and collected by shake off. Whole cell lysates were analyzed by immunoblotting.

Isolated EGFP-FAM110A-WT or -S252-255A proteins were incubated with mock or with active GST-CK1δ in the presence of 32P-γATP for 30 min at 30°C. Phosphorylation of the substrate was detected by a mobility shift in immunoblotting with anti-GFP antibody or by autoradiography. Empty arrowhead indicates the same position on SDS-PAGE gel.

U2OS cells were trapped in mitosis by overnight treatment with nocodazole or PLK1 inhibitor BI2536 (100 nM) and collected by mitotic shake off. Part of the nocodazole arrested cells was further treated with CK1 inhibitor PF-670462 (1 μM) for 60 min. Whole cell lysates were probed with indicated antibodies. Staining for pS10-H3 served as a marker of mitosis.
CK1-mediated phosphorylation of FAM110A promotes its interaction with mitotic spindle and controls chromosomal alignment (2021)
Figure 7

Figure 7. Mitotic function of FAM110A is controlled by its phosphorylation by CK1

U2OS-H2B-EGFP cells were transfected with indicated siRNAs for 48 h or were treated with PF-670462 and filmed in 5 min intervals for 24 h. Time from nuclear envelope breakdown to MAT was quantified in 100 cells. Shown is mean of the medians ± SD (n=3). Statistical significance was determined by ANOVA (**** p<0.0001).

RPE cells or RPE cells stably expressing indicated EGFP-FAM110A variants were transfected with control (siNC) or FMA110A siRNA. Full arrow indicates migration of the EGFP-FAM110A-WT, empty arrow of endogenous FAM110A. Staining for TFIIH was used as a loading control.

RPE cells or RPE cells stably expressing the wild-type or mutant EGFP-FAM110A were transfected with control or two various FAM110A siRNAs and were analyzed by time-lapse microscopy. Plotted are means of the medians of the time from NEB to MAT ±SDs. A hundred cells were quantified per condition (n=3). Statistical significance was determined by ANOVA (**** p<0.0001, ** p<0.005).

RPE cells or RPE cells stably expressing indicated EGFP-FAM110A variants were transfected with control (siNC), FAM110A or CSNK1D siRNA and grown for 48 h. Alternatively, cells were incubated with PF670462 for the last 12 h. Cells were treated with MG132 for 30 min prior fixation. Impaired chromosomal alignment was scored in >30 metaphase cells per condition. Error bars indicate median ±SD. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001, *** p<0.001).

Representative immunoblot analysis of samples from (D). Full arrow indicates migration of the EGFP-FAM110A-WT, empty arrow of endogenous FAM110A.

RPE cells or RPE cells stably expressing indicated mutants were transfected with control or FAM110A siRNAs, fixed after 48 h and stained for γ-tubulin. Metaphase cells were imaged by confocal microscopy (Z-stacks 0.5 μm) and spindle angle between the spindle axis and growth axis was determined. Plotted is a mean of medians from three repeats where 30 cells were imaged for each condition. Error bars indicate median ± SD. Statistical significance was determined ANOVA (n=3) (*** p<0.001, ** p<0.005).

RPE cells or RPE cells stably expressing the wild type FAM110A, -252-255A or -252-255E mutants were transfected with control, FAM110A or CSNK1D siRNAs. Alternatively, cells were treated with PF670462 (1 μM) for 12 h. Spindle angle was determined as in (F). Statistical significance was determined by ANOVA (n=3) (*** p<0.001, ** p<0.005).
CK1-mediated phosphorylation of FAM110A promotes its interaction with mitotic spindle and controls chromosomal alignment (2021)
Figure 8

Figure 8. FAM110A phosphorylation promotes its interaction with microtubules of mitotic spindle

RPE1 were transfected with control siRNA or CSNK1D siRNA or were treated with PF670462 prior fixation. Cells were probed with rabbit polyclonal to FAM110A and mouse monoclonal to γ-Tubulin. Representative images are shown, scale bars indicate 10 μm.

Quantification of (A). Plotted is median FAM110A intensity at spindle poles positive for γ-tubulin ± SD. Each dot represents a single spindle pole. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001).

Cells expressing the wild-type or mutant EGFP-FAM110A were fixed and stained for γ-tubulin and α-tubulin and analyzed by confocal microscopy (z-stack 0.16 µm). Representative images of maximal projections of the metaphase cells are shown, scale bars indicate 10 μm. Merged images show signal of DAPI, γ-tubulin and EGFP. Insets show a single Z plane at the spindle pole.

Quantification of (C). Plotted is median EGFP intensity at spindle poles positive for γ-tubulin ± SD. Each dot represents a single spindle pole. Statistical significance was determined by ANOVA (n=3) (*** p<0.001).

Cells expressing the wild-type FAM110A or FAM110A-S252-255E mutant were treated with DMSO or with PF670462 for 6 h and were stained and imaged as in (C).

Quantification of (E). Plotted is median EGFP intensity at spindle poles positive for γ-tubulin ± SD. Each dot represents a single spindle pole. Statistical significance was determined by ANOVA (n=3) (*** p<0.001, * p<0.05).

HEK293 cells transfected with indicated constructs were treated or not with PF670462 for 6 h. Pull down from cell extracts was performed using GFP trap and binding of tubulin was determined by immunoblotting (n=2).

Expanded View Figure legends
An Organoid-derived Bronchioalveolar Model for SARS-CoV-2 Infection of Human Alveolar-type II-like Cells (2020)
Figure 1

Figure 1 - SARS-CoV-2 infects human organoid-derived 2D small airway cultures.
A, B) Infectious virus titers can be observed by virus titrations on VeroE6 cells of apical washes at 2, 24, 48, and 72h after infection at MOI 0.01 (A) or 0.1 (B) with SARS-CoV-2 (red). The dotted line indicates the lower limit of detection. Error bars represent SEM. N=4. H p.i. = hours post infection.
C-E) Immunofluorescent staining of SARS-CoV-2 infected differentiated small airway cultures. Nucleoprotein (NP) stains viral capsid (red), which colocalized with the ciliated cell marker AcTUB (green; (C, D, E)) and club cell marker CC10 (blue; (D)). Phalloidin was included to stain actin (blue; (C)). Goblet cells are identified by MUC5AC (blue; (E)). Scale bars indicate 50 μm.
An Organoid-derived Bronchioalveolar Model for SARS-CoV-2 Infection of Human Alveolar-type II-like Cells (2020)
Figure 2

Figure 2 - Self-renewing human fetal lung bud tip progenitor organoids differentiate at air-liquid interface to alveolar type I- and II-like cells.
A) Immunofluorescent staining of fetal lung bud tip progenitor organoids grown in expansion medium co-expressing stem cell markers Sox2 (green) and Sox9 (red). Phalloidin (white) was used to stain actin.
B) Immunofluorescent staining of rare HTII-280+ type II pneumocyctes (green) in a subpopulation of fetal lung bud tip progenitor organoids grown in expansion medium.
C) Schematic of the 2D air-liquid interface bronchioalveolar-like model.
D-I) Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express alveolar type I (HOPX, green, (D); HTI-56, green, (E)), type II cells (HTII-280, green, (F); LPCAT1, green, (G); SFTPC (SPC), green, (H)) and basal cell (TP63 (P63), green, (I)) markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium.
Data information: Nuclei are stained with Hoechst (blue in (A, B) or white in (D-I)). Scale bars indicate 50 μm.
An Organoid-derived Bronchioalveolar Model for SARS-CoV-2 Infection of Human Alveolar-type II-like Cells (2020)
Figure 4

Figure 4 - Single cell analysis of bronchioalveolar cultures

A) t-SNE maps showing clustering based on differentially expression analysis and highlighting cell type identification of bronchioalveolar cultures based on cell makers. Each color represents a cluster or different cell type. Number indicates number of cells in cluster identified as cell type presented.
B-K) t-SNE maps highlighting the expression of marker genes for each of the cell types. Transcript counts are given in a Log2 scale.
L) t-SNE map showing clustering based on differentially expression analysis including dataset from A) and published dataset of GSM2855485. Each color represents a single cluster. M) t-SNE map showing clustering of L) with annotation of cell types. Cell type annotation are transferred from A) and published dataset GSM2855485 is annotated as "Purified AT2".Number indicates number of cells in cluster identified as cell type presented.
N, O) t-SNE maps highlighting the expression of SARS-CoV-2 entry receptors ACE2 (N) and TMPRSS2 (O). Transcript counts are given in a Log2 scale.
P) Differentiated bronchioalveolar cultures express SARS-CoV-2 entry receptor ACE2 (green) on ATII-like cells marked by HTII-280 (red). Scale bar indicates 50μm.
Q) Bronchioalveolar cultures co-exress both entry receptors ACE2 (green) and TMPRSS2 (red). Scale bar indicates 50μm.

2.0
An Organoid-derived Bronchioalveolar Model for SARS-CoV-2 Infection of Human Alveolar-type II-like Cells (2020)
Figure 5

Figure 5 - SARS-CoV-2 infects human organoid-derived 2D bronchioalveolar-like cultures.

A, B) Infectious virus titers can be observed by virus titrations on VeroE6 cells of apical washes at 2, 24, 48, and 72h after infection of bronchioalveolar-like cells from donor 1 (A) and donor 2 (B) at MOI 0.01 (red) or 0.1 (black) with SARS-CoV-2. The dotted line indicates the lower limit of detection. Error bars represent SEM. N=4 for all except donor 2 MOI 0.1, which is N=3. H p.i. = hours post infection.

C-E) Immunofluorescent staining of SARS-CoV-2 infected differentiated bronchioalveolar-like cultures. Nucleoprotein (NP) stains viral capsid (red), which colocalized with HOPX (C), HTII-280 (D) and SFTPC (E). Scale bars indicate 50 μm.
An Organoid-derived Bronchioalveolar Model for SARS-CoV-2 Infection of Human Alveolar-type II-like Cells (2020)
Figure 6

Figure 6 - Host response analysis of SARS-CoV-2 infection in organoid-derived bronchioalveolar-like and small airway cells.

A) pSTAT1 translocation in mock and SARS-CoV-2-infected bronchioalveolar-like cells visualized in green. Scale bars indicate 50 μm. Nucleoprotein (NP) stains viral capsid (red). Nuclei are stained with Hoechst (white).

B) Heatmaps depicting top 20 enriched genes based on adjusted pvalue in bronchioalveolar-like cells after SARS-CoV-2 infection. Colored bars represent Z-scores of log2 transformed values.

C) pSTAT1 translocation in mock and SARS-CoV-2-infected small airway cells visualized in green. Nucleoprotein (NP) stains viral capsid (red). Nuclei are stained with Hoechst (white). Scale bars indicate 50 μm.

D) Heatmaps depicting top 25 enriched genes based on adjusted pvalue and log2 fold changes in small airway cells after SARS-CoV-2 infection. Colored bars represent Z-scores of log2 transformed values.

E) GO term enrichment analysis for biological processes of the 20 most significantly up-regulated genes upon SARS-CoV-2 infected in bronchioalveolar-like cells. P-values were determined using PANTHER Overrepresentation Test.

F) GO term enrichment analysis for biological processes of the 25 most significantly up-regulated genes upon SARS-CoV-2 infected in small airway cells. P-values were determined using PANTHER Overrepresentation Test.
An Organoid-derived Bronchioalveolar Model for SARS-CoV-2 Infection of Human Alveolar-type II-like Cells (2020)
Figure 7

Figure 7 - Low dose interferon lambda 1 treatment dramatically reduces SARS-CoV-2 replication in bronchioalveolar-like cultures.
(A, B) SARS-CoV-2 replication shown as infectious virus titers (A) and viral RNA copies (B) in apical washes at 2, 24, 48, and 72h after infection at MOI 0.1. Cultures were either pretreated with 50 ng/ml IFN lambda 1 in the basal compartment two hours prior to the infection (green), at 24 h p.i. or left untreated. The dotted line indicates the lower limit of detection. Error bars represent SEM. N=2. H p.i. = hours post infection.

C) Whole well immunofluorescent staining of representative cultures in (A) and (B) using an anti-NP antibody (red). Nuclei are stained with Hoechst (blue). Scale bar indicates 1mm.
Rab11-FIP1 mediates Epithelial-Mesenchymal Transition and invasion in esophageal cancer (2020)
Figure 1

Figure 1. Mutant p53 increases tumorigenic potential of ESCC cells.

A. Schematic of the p53 mouse model which generates L2-Cre;Trp53+/+;R26lox-Stop-lox-YFP, L2-Cre;Trp53-/-;R26lox-Stop-lox-YFP and L2-Cre;Trp53R172H/- ;R26lox-Stop-lox-YFP mice. Mice were treated with 4NQO in the drinking water for 16 weeks followed by normal water. Esophageal epithelial cells were isolated and cell lines were generated.

B. Cell lines generated from WT, null, and mutant p53 esophagi were assessed by Western blot analysis for p53 protein expression. Relative intensity densitometry of Rab11-FIP1 results are shown at the bottom.

C. Organoids generated from WT, mutant, and null p53 cell lines. Scale bar: 100μm.

D. Quantification of the portion of organoids exhibiting a solid, intermediate or stratified morphology (> 37 organoids from 2-3 cell lines per group).

E-G. Cells were subcutaneously injected in the lower flanks of nude mice (n= 4 flanks/cell line. WT p53 2 cell lines, Mutant p53-3 cell lines, p53 null-3 cell lines). E. The number of tumors formed in each group was quantified. F. Tumor growth was measured at the indicated time points post-implantation. Graph represents total tumor size per group. G. Average tumor weight following dissection (n=2 biological replicates for WT p53 cell lines, n=3 biological replicates for mutant p53 cell lines, n=3 biological replicates for p53 null cell lines, n=4 technical replicates injected per cell line, calculated as average tumor weight per cell line). Error bars represent ±S.E.M. *p=0.02 Mutant p53 vs WT p53, *p=0.02 Mutant p53 vs Null p53 (One-way Anova).