EMBO/soda-nel huggingface dataset:
Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex (2019)
Figure 5

Figure 5. Increased Mad1-Bub1 interaction bypasses the requirement for Rod

A) A HeLa cell line stably expressing Mad1-BirA was depleted of Rod and synchronized in mitosis using thymidine and nocodazole. Biotin was added to media where indicated and biotinylated proteins were purified and analyzed by western blot with the indicated antibodies. Representative of 2 independent experiments.

B) The duration from NEBD to exit in each indicated conditions was recorded by time-lapse microscopy. Mean (red line) and standard error of mean (black bar) indicated. (Mann Whitney test, ****: P ≤ 0.00001). A representative result from at least 3 independent experiments is shown.

C) Mitotic duration in the indicated conditions expressing Mad1-Bub1 (Mad1 485-718 fused to Bub1 1-553ΔCD1) fusion protein. The time from NEBD to exit is indicated. Red circles represent cells still arrested in mitosis when the filming stopped. Mean (red line) and standard error of mean (black bar) indicated. (Mann Whitney test, ns: non-significant, ****: P ≤ 0.00001). A representative result from at least 3 independent experiments is shown.

D, E) Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. (D) Representative immunofluorescence images of cells stained for YFP and Mad1 in each condition. Scale bar, 5 μm. (E) Mad1 kinetochore levels were measured and normalized to Bub1 (YFP) level. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.
Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex (2019)
Figure 6

Figure 6. Analysis of Bub1 4xCD1 in Bub1 CR and parental cells.

A) Bub1 CR cells complemented with the indicated Bub1 constructs and time from NEBD to exit in nocodazole measured by time-lapse microscopy. Mean (red line) and standard error of mean (black bar) indicated. (Mann Whitney test, ns: non-significant, ****: P ≤ 0.00001). A representative result from at least 3 independent experiments is shown.

B) Kinetochore levels of Mad1 in Bub1 CR cells complemented with the indicated YFP tagged Bub1 constructs. Mad1 levels are normalized to YFP signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown.

C, D) HeLa cells transfected with the indicated Bub1 constructs and time from NEBD to exit determined during an unperturbed mitosis. (C) Still images of unperturbed mitosis of HeLa cells transfected with Venus-Bub1 4xCD1. CFP-Histone 3 was used as the chromosome marker (the middle panel) and Venus-Bub1 4xCD1 was shown in the bottom panel. Scale bar, 5 μm. (D) The time from NEBD to exit was analyzed in each condition. Mean (red line) and standard error of mean (black bar) indicated. (Mann Whitney test, ns: non-significant). A representative result from at least 3 independent experiments is shown.
Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex (2019)
Figure 7

Figure 7. Bub1 recruitment of RZZ to kinetochores.

A) Localization of Venus-Rod during unperturbed mitosis in control depleted or Bub1 depleted cells.

B) Primary structure of Bub1 with sequence of CD1 shown and residues mutated in red. Schematic of fusion constructs used in panels (E and F).

C) HeLa cells were depleted of Bub1 using RNAi and complemented with indicated Venus-Bub1 constructs. Immunofluorescence images were shown for Bub1 and ZW10 localization.

D) Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions.

E, F) As in (C and D) but with the indicated Bub1 fusion proteins (Student's t-test used for statistical comparison, ns: non-significant, ****: P ≤ 0.00001).

Data information: Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown. Scale bars, 5 μm.

Table 1

Analysis of mitotic timing and segregation errors during an unperturbed mitosis.

Expanded View FIGURE LEGENDS
Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells (2022)
Figure 1

Figure 1: A genome-wide CRISPR screen for SARS-CoV-2 entry host factors.

A) Infection susceptibility (top) to VSVG and Spike-typed lentiviruses of a panel of lung cancer cell lines with or without (Empty vector) overexpression of ACE2. ACE2 protein levels were determined by Western-blot (bottom).

B) Screen strategy: Calu1ACE2 cells were transduced with the GeckoV2 CRISPR library to create a mutant library that was subsequently infected with Spike-typed lentiviruses carrying a hygromycin resistant plasmid. After hygromycin selection, dropout of sgRNAs in the infected population compared to an uninfected population was assessed by NGS.

C) Gene-level scores obtained by robust ranking algorithm analysis of the sgRNA relative abundances between cells infected with Spike-typed lentiviruses and uninfected cells. Genes were ordered alphabetically in the x-axis and the top hits (log10(enrichment-score) < -5) were colored and labelled. Note that CTSL and CTSL1 are different gene isoforms.

D) Screen results integration in pathways previously linked to SARS-CoV-2 entry. Black: non-significant genes reported by others; Blue: significant genes previously reported; Red: novel genes not reported in previous screens. * Non-significant based on FDR (FDR > 0.05) but present in the top 200 hit list.
Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells (2022)
Figure 2

Figure 2: Validation of screen results with pseudotyped lentiviruses.

A) Scheme depicting the screen validation strategy (top) and representative density plots (bottom) of flow cytometry data from cells infected with VSVG- or Spike-typed lentivirus. The infected population is labeled with a darker color.

B) Susceptibility to infection with Spike-typed and VSVG-typed lentiviruses in Calu1ACE2 CRISPR KO cell lines with loss-of-function of selected screen candidates. NT596 and NT764 are two non-targeting CRISPR controls. ACE2 are Calu1ACE2 cells transduced with a CRISPR vector against ACE2. Bars represent the average and standard error of the mean (SEM) of the percentage of infected cells in each condition (three biological replicates), normalized to the non-targeting control cell line NT596. The t-test p-value between the relative percentage of infection with Spike-typed and VSVG-typed for each cell line is indicated.

C) Confocal microscopy images of SPNS1-KO1 and PLAC8-KO1 Calu1ACE2 cells overexpressing CRISPR-resistant SPNS1- or GFP-PLAC8, respectively. Scale bar: 20 µm.

D) Rescue experiments in PLAC8 and SPNS1 Calu1ACE2 KO cell lines: bar plot showing the average (three biological replicates) and SEM percentage of infection (normalized to NT596 with GFP overexpression) of Spike-typed and VSVG-typed lentiviruses in PLAC8-KO1 and SPNS1-KO1 cell lines that overexpress CRISPR-resistant PLAC8- or GFP-SPNS1 constructs. The significance above each bar represents the t-test p-value between each condition and the control cell line (NT596 with GFP overexpression)

Data information: ns, p-value >= 0.05, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001.
Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells (2022)
Figure 3

Figure 3: Validation of screen results using full SARS-CoV-2 infectious viruses.

A, B) Susceptibility to infection with SARS-CoV-2 viruses in Calu1ACE2 (A) and H1299ACE2 (B) cell lines with loss-of-function of the indicated genes. Left: representative immunofluorescence images of SARS-CoV-2 nucleocapsid protein (green) and cell nuclei (red) in the indicated cell lines. Scale bar: 20 µm. Right: immunofluorescence quantification of the number of infected cells at 24 h post-infection in the different loss-of-function cell lines infected with SARS-CoV-2. Bars represent the average and SEM of the percentage of infected cells relative to CRISPR non-targeting transduced (NT596) cells in three biological replicates (three different CRISPR KO cell lines infected independently). The significance above each bar represents the one-tail t-test p-value between each condition and CRISPR non-targeting transduced (NT596) cells.

C) Western-blot analyses of SPNS1 and PLAC8 protein levels in the different lung cancer cell lines tested in Fig 1 with or without ACE2 overexpression. Note that PLAC8 and SPNS1 immunodetection was performed on the same blots than in Figure 1A, and that ACE2 and Actin panels from Figure 1A were included here to allow comparison.

D) Susceptibility to Spike-typed lentiviruses in the indicated ACE2 overexpressing cancer cell lines that were transduced with GFP, GFP-PLAC8 or GFP-SPNS1. Bars represent the average and SEM of the percentage of infected cells in three technical replicates (independent infections of the same cell line) relative to Calu1ACE2-GFP cells. The significance above each bar represents the t-test between GFP-PLAC8 or GFP-SPNS1 and GFP cells for each cell line.

E) Susceptibility to infection with SARS-CoV-2 viruses in cell lines that overexpress ACE2 and GFP-SPNS1 or GFP-PLAC8. Bars represent the immunofluorescence quantification of the number of SARS-CoV-2 infected cells at 24 h post-infection. Representative images of SARS-CoV-2 nucleocapsid immunofluorescence (green) and cell nuclei (red) in GFP and GFP-PLAC8 cells are shown right to each plot. Scale bar: 20 µm.

Data information: ns, p-value >= 0.05, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001.
Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells (2022)
Figure 5

Figure 5: PLAC8 and SPNS1 do not affect binding and endocytosis.

A) Binding and endocytosis studies with Alexa Fluor 647-conjugated S-RBD or transferrin in Calu1-ACE2-RFP cells deficient in PLAC8 and SPNS1. Left: scatterplots of flow cytometry data showing the cutoffs used to define ACE2-RFP and Alexa Fluor 647 positive cells. Right: bar plots showing the ratio of cells positive for Alexa Fluor 647 in either ACE2-RFP positive (red) or negative (blue) cells in binding and endocytosis experiments with S-RBD or transferrin. Bars represent the average and SEM of three biological replicates (three different CRISPR KO cell lines). The significance above each bar represents the t-test p-value between each condition and CRISPR non-targeting transduced (NT596) cells. ns, p-value >= 0.05, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001.

B-E) Endocytosis experiments with S-RBD in PLAC8- or SPNS1-KO Calu1-ACE2-RFP cells with overexpression of GFP-PLAC8 or GFP-SPNS1, respectively. Cells were incubated with S-RBD for 1 h at 37 ºC and fixed with 4% p-formaldehyde after an acid wash to remove non-internalized S-RBD. Representative wide-field fluorescence images (B) and quantification of mean fluorescence intensities of each fluorophore (C) in each cell show that S-RBD signal correlates with ACE2-RFP expression but not with GFP-PLAC8 or GFP-SPNS1. The Pearson correlation coefficient (r) and correlation test p-value (p) is indicated within each scatterplot. Each dot represents a cell measurement from different microphotographs of a single replicate. Scale bar: 20 µm. Representative fluorescence confocal microphotographs (D) and spot colocalization analyses using ComDet plugin (E) demonstrate colocalization of S-RBD endosomes with ACE2-RFP and GFP-PLAC8 or GFP-SPNS1. (D) Red ovals defining the detected S-RBD dots are overlaid in each channel to assess the colocalization with ACE2-RFP, GFP-PLAC8 or GFP-SPNS1. Inlets show a magnification of the region defined by the white squares. Scale bar: 20 µm. (E) Boxplots representing the percentage of internalized S-RBD that colocalize with ACE2-RFP, GFP-PLAC8, GFP-SPNS1 or their combination (ACE2-RFP and GFP-PLAC8 or ACE2-RFP and GFP-SPNS1). The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate.
Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells (2022)
Figure 6

Figure 6: PLAC8 and SPNS1 deficiency increases autophagic activity.

A) Venn diagrams showing the overlap of significantly up- or down-regulated proteins in mass spectrometry studies between PLAC8- and SPNS1-KO Calu1ACE2 cells, compared to CRISPR non-targeting control cells (NT596).

B) Dot plot summarizing the results of gene set enrichment analyses (GSEA) in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596). The size of the dots indicates the number of proteins in each category, while the color scale indicates the false discovery rate (FDR) values.

C) Representative images (left) and quantification (right) of the number of acidic particles in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596) using live-cell lysotracker staining. The two samples Wilcoxon Test p-value of the pooled observations compared to NT596 cells is indicated above each condition. The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate. Three biological replicates per condition were used. Scale bar: 20 µm

D) Lysosomal degradation studies in Calu1ACE2 cells using the fluorogenic substrate DQ-Red-BSA. Left: density plots representing the shift in fluorescence intensity in NT596 cells untreated or treated with DQ-Red-BSA under growth or serum starvation conditions. Right: bar plot representing the mean and SEM of the median fluorescence intensity values (MFI) of three biological replicates of PLAC8- and SPNS1-KO and CRISPR non-targeting cells in growth and serum starvation conditions.

E) Representative images (left) and quantification (right) of the number of LC3B particles in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596) using LC3B immunofluorescence staining. The two samples Wilcoxon Test p-value of the pooled observations compared to NT596 cells is indicated above each condition. The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate. Three biological replicates per condition were used. Scale bar: 20 µm

F) Autophagic flux analyses using the fluorescence reporter GFP-LC3-RFP-LC3ΔG in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596). Left: density plots showing the shift in GFP intensity under starvation conditions compared to control growth conditions in NT596 Calu1ACE2 cells. Right: bar plots showing the mean and SEM of the median GFP/RFP intensity ratio of three biological replicates in the different cell lines and conditions.
Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors (2022)
Figure 5

Figure 5: The effect of various mutants on the MS- and C-complexes of H. pylori fliP*.

A-G) Slices through electron cryotomograms (left panels), central slices of subtomogram averages (middle panels), and schematic representations (right panels) of the MS- and C-complexes (white arrows on the slices) of different mutants in H. pylori fliP*. Light and dark green arrows in (A and D) point to the ~53-nm and ~67-nm cytoplasmic rings. Scale bars for cryotomogram slices are 50 nm, and 20 nm for STAs. Empty panels indicate that there were not enough examples to produce a STA.
The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures (2020)
Figure 1

Figure 1. CBF1 promotes Arabidopsis hypocotyl elongation in the light.

Phenotypes of 4-d-old Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown at 22°C in darkness (D) or continuous W, R, FR, and B light. Scale bar = 1 mm.

Hypocotyl lengths of Col, two cbf1 mutants and two CBF1-OE lines grown at 22°C in D or continuous W, R, FR, and B light. Error bars represent SD from 20 seedlings. *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col. NS, not significant.
The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures (2020)
Figure 2

Figure 2. CBF1 transcript and protein levels are induced by light.

qRT-PCR analysis showing the relative expression of CBF1 in 4-d-old wild-type (Col) seedlings grown at 22°C in darkness (D) or continuous W, R, FR, and B light. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).

GUS staining of 4-d-old homozygous CBF1p:GUS transgenic seedlings grown at 22°C in D or continuous W, R, FR, and B light. Scale bar = 1 mm.

qRT-PCR data showing that CBF1 expression is induced by different light regimes. Wild-type (Col) seedlings were first grown at 22°C in D for 4 d, and then transferred to continuous W, FR, R, or B light for the indicated times ranging from 1 h to 24 h. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).

Immunoblots showing CBF1 protein levels in nuclear fractions from 4-d-old wild-type (Col) seedlings grown at 22°C in D or continuous W, R, FR, and B light.

Immunoblots showing CBF1-myc protein levels in 4-d-old CBF1-myc seedlings grown at 22°C in D or continuous W, R, FR, and B light.

F, G Immunoblots showing the levels of CBF1-myc proteins after exposure to white light. CBF1-myc seedlings were first grown at 22°C for 4 d in D, and then transferred to continuous W light for the indicated time periods within 1 h (F) or ranging from 1 h to 24 h (G).

Immunoblots showing the levels of CBF1-myc proteins after exposure to FR (H), R (I) or B (J) light. CBF1-myc seedlings were first grown at 22°C for 4 d in D, and then transferred to continuous FR, R or B light for the indicated time periods ranging from 1 h to 24 h.

Data information: In (D), Anti-H3 was used as a sample loading control. In (E) to (J), anti-HSP was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1-myc normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot.
The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures (2020)
Figure 3

Figure 3. CBF1 physically interacts with phyA and phyB.

In vitro pull-down of PHYA-C1 (A) and PHYB-C1 (B) with CBF1. The His-tagged PHYA/B-N (photosensory domain), PHYA/B-C1 (PAS-related domain), and PHYA/B-C2 (histidine kinase-related domain) proteins pulled down with GST-tagged CBF1 or GST were detected by anti-His antibody. Input, 6% of the His-tagged purified target proteins used in pull-down assays.

LCI assays showing that CBF1 interacted with phyA in living plant cells. Scale bar = 1 cm.

D, E Co-IP assays showing that CBF1 interacted with phyA (D) and phyB (E) in vivo. (D) homozygous CBF1-myc seedlings were first grown at 22°C in continuous FR light for 4 d, then the total proteins were extracted and subsequently treated with 5-min R light, or 5-min R light followed by 5-min FR light (R+FR) before immunoprecipitation. (E) homozygous CBF1-myc seedlings were first grown at 22°C in continuous R light for 4 d, then the total proteins were extracted and subsequently treated with 5-min FR light, or 5-min FR light followed by 5-min R light (FR+R) before immunoprecipitation. After light treatments, total proteins were incubated with anti-c-myc Affinity Gel (Sigma-Aldrich). The total and precipitated proteins were examined by immunoblotting using antibodies against myc, phyA, phyB, and RPN6, respectively. The numbers below anti-phyA and anti-phyB blots indicate the relative band intensities of co-precipitated phyA or phyB normalized to those of precipitated CBF1-myc, respectively. The ratio of the first clear band was set to 100 for each blot.
The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures (2020)
Figure 4

Figure 4. CBF1 promotes PIF4 and PIF5 protein accumulation in the light.

Immunoblots showing the levels of PIF4 and PIF5 proteins at 22°C in 4-d-old Col, cbf1-1, cbf1-2, and CBF1-myc seedlings grown in darkness (D) or continuous W or R light. Anti-RPN6 was used as a sample loading control.

Phenotypes and hypocotyl lengths of 4-d-old Col, cbf1-1, pif4 pif5, cbf1 pif4 pif5, 35S:PIF4 and 35S:PIF4 cbf1 seedlings grown at 22°C in continuous W or R light. Error bars represent SD from 20 seedlings. **P < 0.01 and ***P < 0.001 (two-tailed t-test) for the indicated pairs of seedlings. Scale bar = 1 mm. NS, not significant.

Immunoblots showing the PIF4 protein levels in 4-d-old Col, 35S:PIF4, and 35S:PIF4 cbf1 seedlings grown at 22°C in D or continuous W or R light. Anti-RPN6 was used as a sample loading control.

Data information: In (A) and (C), numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of RPN6, respectively. The ratio of the first clear band was set to 100 for each blot.
The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures (2020)
Figure 5

Figure 5. CBF1 inhibits phyB interaction with PIF4 and PIF5.

Yeast three-hybrid assays showing that CBF1 represses the interaction of the Pfr-phyB with PIF4 (A) and PIF5 (B) in yeast cells. AD-PIF4, AD-PIF5, phyB-BD and CBF1 were expressed in the yeast strain Y190 as indicated. The β-galactosidase activities were measured by liquid culture assays using ONPG as the substrate. Error bars represent SD of three independent yeast clones.

C, D LCI assays showing that CBF1 inhibits phyB interaction with PIF4 (C) and PIF5 (D) in plant cells. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).

E, F Semi-in vivo pull-down assays showing that CBF1 inhibits phyB interaction with PIF4 (E) and PIF5 (F). The total proteins extracted from 4-d-old 35S:phyB-GFP seedlings grown at 22°C in continuous R light served as the bait, and GST-PIF4 or GST-PIF5 fusion proteins served as the prey. Equivalent amounts of phyB-GFP protein extract and GST-PIF4 or GST-PIF5 fusion proteins, and increasing amounts of His-CBF1 were added as indicated before anti-GFP IP assays were performed. The pulled-down proteins were subjected to immunoblotting with antibodies against phyB, GST and His, respectively. Numbers below the anti-GST blots indicate the relative band intensities of co-precipitated GST-PIF4 or GST-PIF5 normalized to those of precipitated phyB-GFP, respectively. The ratio of the first clear band was set to 100 for each blot.