EMBO/soda-nel huggingface dataset:
Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition (2021)
Figure 4

Figure 4 Phf5a depletion impairs NHEJ.

(A) Schematic diagram of the reporter construct for analyzing the DNA DSB repair by NHEJ. Incompatible DNA ends produced by I-SceI-induced DNA cleavage are repaired by NHEJ, which removes the intervening TK, resulting in GFP expression. Triangles indicate the positions of primers used to amplify a fragment encompassing the repaired junction after I-SceI cleavage.

(B) Effect of Phf5a knockdown on I-SceI-induced NHEJ. Plot represents the GFP-positive cells obtained by FACS analysis after co-transfection of the I-SceI expression plasmid and the indicated siRNAs into a cell line harboring the NHEJ reporter construct. The values are presented as mean ± sd (n=3). Statistical significance was evaluated against corresponding siControl by two-tailed unpaired Student's t-test (*** p ≤0.001).

(C) Genomic DNA was isolated from each transfectant and subjected to PCR using the primer pair indicated in A.

(D) Confirmation of Phf5a KD in the samples by qRT-PCR. The values are presented as mean ± sd (n=3). Statistical significance was evaluated against corresponding siControl by two-tailed unpaired Student's t-test (*** p ≤0.001).

(E) Representative NHEJ junctions obtained from siControl- and siPhf5a-treated samples.
Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition (2021)
Figure 5

Figure 5 Impaired recruitment of NHEJ factors to the IgH locus upon Phf5a deficiency. (A) Analysis of Sμ-Sα recombination junctions in the genomic DNA isolated from siControl- and siPhf5a-transfected CH12F3-2A cells stimulated by CIT for 48 h. The values are presented as mean ± sd (n=3), Statistical significance was evaluated in reference to siControl by two-tailed unpaired Student's t-test (*p ≤ 0.05, ** p ≤0.01, *** p ≤0.001).

(B) The proportion of insertions versus microhomology regions of various lengths in the Sμ-Sα junctions presented as a pie chart. The number of clones analyzed from control and Phf5a-depleted samples are indicated at the center.

(C) Top: schematic diagram of the position of the ChIP assay PCR products specific to the S region. Bottom: ChIP assay of the indicated DNA repair proteins using CIT-stimulated CH12F3-2A cells transfected with siControl or siPhf5a. The values are presented as mean ± sd (n=3). Statistical significance was evaluated in reference to siControl by two-tailed unpaired Student's t-test (*p ≤ 0.05, ** p ≤0.01, *** p ≤0.001).
Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition (2021)
Figure 6

Figure 6 Knockdown of Phf5a alters the H2A variant deposition at the S region.

(A) Schematic illustration of the Sμ and Sα regions showing the position of the ChIP PCR products.

(B) ChIP analysis of H2A and its variants using CIT-stimulated CH12F3-2A cells transfected with siControl or siPhf5a. The antibodies used for each ChIP set are indicated at right. The values are presented as mean ± sd (n=3). Statistical significance was evaluated in reference to the siControl by two-tailed unpaired Student's t-test ( *p ≤ 0.05, ** p ≤0.01).

(C) Western blot analysis of the whole cell extract from CIT-stimulated CH12F3-2A cells transfected with the indicated siRNAs. Expression of various proteins including AID, variant H2As and Ku80 were not decresed by Phf5a KD. (ns, non-specific)
Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition (2021)
Figure 7

Figure 7 The Phf5a mutants with a loss of CSR function show defects in protein-protein interactions.

(A) Amino acid sequence of mouse Phf5a protein. The 12 Cys residues indicated in red are part of the PHD finger-like domain. Each CxxC motif is underlined and color coded according to their pairing with another CxxC motif to form a zinc finger (ZF). A pair of CxxC motifs hold the zinc atom in each ZF. Arrangement of 4 Cys residues in ZF1-3 are schematically shown below the Phf5a sequence.

(B) CH12F3-2A cells were transfected with siRNAs along with empty vectors or vectors expressing various zinc finger mutants of Phf5a (Phf5aR-MF). Twenty-four hours after transfection, the cells were stimulated by CIT for another 24 h, and then examined for CSR. The efficiency of IgA switching was calculated relative to the value obtained for siControl and empty vector-transfected cells. The data are compiled from three independent experiments. The values are presented as mean ± sd (n=3). Statistical significance was performed in reference to siControl by two-tailed unpaired Student's t-test ( *** p ≤0.001). See Supplementary Fig.S8 for a representative data set.

(C) CH12F3-2A cells were transfected with WT Phf5a or its mutants (Phf5aR-MF) along with siRNA against Phf5a. An anti-Flag antibody was used to immunoprecipitate and immunodetect Phf5aR-MF. An anti-Phf5a immunoblot confirmed the knockdown efficiency of the endogenous Phf5a. The antibodies used to detect the co-associated proteins are indicated next to the respective blot.

(D) CH12F3-2A cells transfected with WT or a mutant Phf5a (Phf5aR-MF) were subjected to anti-p400 immunoprecipitation. Associations with relevant interacting proteins were examined by immunoblot analysis, and are indicated next to each blot. (E) ChIP assay derived from CH12F3-2A cells transfected with the indicated siRNA and Phf5a (Phf5aR-MF) expression constructs. Antibodies used for ChIP are indicated on the top of the respective plot. The data are summarized from 2 independent experiments and presented relative to siControl treated sample, which was set as 100. The error bars represent the mean ± sd.
Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition (2021)
Figure 8

Figure 8 Phf5a zinc finger-2 (ZF2) plays an important role in CSR and epigenomic regulation required for DNA repair.

(A) A representative FACS profile of CSR complementation assay using WT and mutant Phf5 as indicated and describe in Fig 7B.

(B) Western blot showing expression of endogenous and exogenously introduced Phf5a.

(C) H2A.Z ChIP assay was performed using CH12F3-2A cells transfected with indicated siRNA and the Phf5a expression constructs. The data are compiled from 2 experiments and presented relative to siControl treated sample, which was set as 100. The error bars represent the mean ± sd.

(D) The schematic illustration of Phf5 zinc fingers (ZF1-3) was adapted from van Roon et al., 2008. The Cys75 and Cys72 are indicated by thick and thin arrows, respectively. (E) Illustration depicting AID-induced DNA breaks and repair in the S region, which leads to CSR and IgH/c-Myc chromosomal translocation. The scheme also highlights the involvement of the Phf5a-p400 pathway, which promotes H2A.Z and H2AX deposition in the recombining S regions and facilitates NHEJ-mediated DNA repair.
BRD9 is a Druggable Component of Interferon-Stimulated Gene Expression and Antiviral Activity (2021)
Figure 3

Figure 3. Targeted Degradation of BRD9 Reveals its Cell-Type Independent Contribution to Interferon-Stimulated Antiviral Activity Against Multiple Viruses.

A. Schematic representation of the mechanism of action of dBRD9-A. dBRD9-A simultaneously recruits the ubiquitin E3 ligase Cereblon (CRBN) to the bromodomain (BD) of BRD9, thereby facilitating the ubiquitination and degradation of BRD9.

B. A549 cells were treated for the indicated time with either DMSO or 125nM dBRD9-A. Following lysis and western blot analysis for the indicated proteins, BRD9 levels were quantified by densitometry and made relative to levels in DMSO-treated cells (% below). Data are representative of at least two biological replicates.

C. A549 cells were treated with 125nM dBRD9-A (+) or DMSO (-) for 6h prior to lysis and analysis by western blot for the indicated proteins. Data are representative of at least two biological replicates.

D. A549 cells were treated with 125nM dBRD9-A (+) or DMSO (-) for 6h prior stimulation with 1000 IU/mL of IFN-α2 for 16h. Following lysis, western blot was used to detect levels of the indicated proteins. Data are representative of at least two biological replicates.

E. A549 cells were treated as described in (D), but following stimulation with 1000 IU/mL of IFN-α2 for 16h were infected with IAV (WSN/33) at an MOI of 0.01 PFU/cell. Viral titers were determined after 24h by plaque assay. Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed plaque counts (***p-value < 0.001).

F-H. A549 cells were treated as described in (E), but IFN-α2 concentrations ranged from 10-1000 IU/mL depending upon the virus, and infections were similarly performed but with: VSV-GFP (F); HSV1-GFP (G); or VSV-G pseudotyped HIV1-GFP (H).

I-L. Hep2 (I), Huh-7 (J), MRC-5 (K), and undifferentiated HTBE (L) were treated and infected with VSV-GFP as in (F).

Data information: For F-L, Total Integrated Green Fluorescent Intensities were determined using the Incucyte live-cell analysis system after 10h (G), 24h (F, I-L) or 48h (H). Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed intensity values (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001). For E-L, dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions. See also Figure EV3.
BRD9 is a Druggable Component of Interferon-Stimulated Gene Expression and Antiviral Activity (2021)
Figure 4

Figure 4. BRD9 Promotes the Interferon-Stimulated Expression of a Subset of Antiviral Genes.

A. Overview of the dBRD9-A transcriptomics experiment in A549 cells.

B. Up- (red) and down- (blue) regulated genes identified in A549 cells following treatment with 100 IU/mL IFN-α2 for 6h: ISGs were defined as induced by IFN-α2 with FC>2, p-value ≤ 0.001.

C. Impact of dBRD9-A pre-treatment on the induction of ISGs identified in (B). Colors indicate IFN-induced transcripts significantly affected (blue) or unaffected (gray) by dBRD9-A pretreatment (FC>2, p-value ≤ 0.001).

D. Heatmaps highlighting the gene expression changes observed with each treatment. Left: the top 50 ISGs, ranked by IFN-induced expression (B), were compared with respect to the impact that dBRD9-A pre-treatment had on induction. Right: comparative fold change in induction for each ISG in the presence or absence of dBRD9-A.

E. ISG expression level following IFN-α2 stimulation, grouped by whether they were affected or not by dBRD9-A pretreatment (see C).

F. RT-qPCR confirmation of transcriptomics results for selected transcripts (MX1, MX2, IFITM1, ISG15 and IFIT1) in A549 cells treated as outlined in (A). GAPDH transcript levels were used for normalization. Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by unpaired 2-tailed t-test on ΔCt values (*p-value < 0.05; n.s. not significant).

Data information: See also Figure EV4.
BRD9 is a Druggable Component of Interferon-Stimulated Gene Expression and Antiviral Activity (2021)
Figure 5

Figure 5. BRD9 Function Requires its Acetyl-Binding Activity and Unique DUF3512 Scaffolding Domain.

A. Top: schematic representation of wild-type (wt) human BRD9 and selected mutants, highlighting the acyl-binding bromodomain (BD9) and the DUF3512 domain responsible for ncBAF assembly. Numbers represent amino-acid residues. The box highlights BD9 positions 208-216, which include residues critical for acetyl (N216) or butyryl (M208) binding. dBD is an engineered BRD9 variant lacking the entire bromodomain (residues 140-237). Bottom: western blot analysis of CTRL#3 or BRD9-KO#3 cells stably transduced with lentiviruses expressing empty vector (EV), or wt and mutant BRD9 constructs.

B. The various CTRL#3 or BRD9-KO#3 cells described in (A) were stimulated with 1000 IU/mL of IFN-α2 for 16h prior to infection with VSV-GFP at an MOI of 0.6 PFU/cell. Total Integrated Green Fluorescent Intensities were determined using the Incucyte live-cell analysis system at 48h post-infection.

C. Schematic representation of HA-tagged wt human BRD9, BRD7, and selected chimeric mutants. BD7 is the BRD7 bromodomain. Numbers represent amino-acid residues of the final constructs.

D. Western blot analysis of CTRL#3 or BRD9-KO#3 cells stably transduced with lentiviruses expressing empty vector (EV), or HA-tagged wt and chimeric mutant constructs described in (C).

E. The various CTRL#3 or BRD9-KO#3 cells described in (D) were stimulated and infected as described in (B).

F. Schematic representation of wt human BRD9, and selected chimeric mutants with domains from BRD7. Numbers represent amino-acid residues of the final constructs.

G. Western blot analysis of CTRL#3 or BRD9-KO#3 cells stably transduced with lentiviruses expressing empty vector (EV), or wt and chimeric constructs described in (F).

H. Western blot analysis of input lysates and anti-BRD9 immunoprecipitations (IPs) from cells described in (G) expressing wt BRD9 or the chimeric BRD7DUF. The indicated proteins were detected with specific antibodies. Data are representative of two biological replicates.

I. The various CTRL#3 or BRD9-KO#3 cells described in (G) were stimulated and infected as described in (B).

Data information: For (B), (E) and (I), data represent means and standard deviations from n=3 biological experiments (individual data points shown). Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions. Dotted lines are a visual guide for minimum virus replication in control cells in the presence of IFN-α2. Statistical significance was determined by 1-way ANOVA on log-transformed intensity values (***p-value < 0.001; ****p-value < 0.0001; n.s. not significant). See also Figure EV5.
The role of interleukin-6 in monitoring severe case of coronavirus disease 2019 (2020)
Figure 1

Figure 1. Laboratory findings in severe versus non-severe COVID-19 patients.

A) Neutrophils and lymphocytes count.

B) Levels of creatine kinase (CK) and creatinine.

C) Levels of procalcitonin (PCT) and C-reactive protein (CRP).

D) Levels of erythrocyte sedimentation rate (ESR) and ferritin.

E) Levels of D-dimer and lactate dehydrogenase (LDH).

F) Proportion of total CD3+ and CD4+ T cells.

G) Proportion of CD8+ T cells and CD4+/CD8+ T cell ratio.

H) Proportion of B and natural killer (NK) cells.

I-K) cytokine profile of COVID-19 patients for IL-2, IL-4 (I), IL-6, IL-10 (J) and TNF-α and IFN-γ (K).

Data information: Statistical analysis was performed by paired two-tailed Mann-Whitney U test (A-D). Blue dotted lines denote normal value or normal range. Error bars, SEM.
The role of interleukin-6 in monitoring severe case of coronavirus disease 2019 (2020)
Figure 3

Figure 3. Correlation between baseline IL-6 level and clinical and laboratory characteristics of severe COVID-19 patients.

Correlation between IL-6 and maximal body temperature during hospitalization.

Correlation between IL-6 and C-reactive protein (CRP).

Correlation between IL-6 and lactate dehydrogenase (LDH).

Correlation between IL-6 and D-dimer.

Correlation between IL-6 and ferritin.

Levels of baseline IL-6 in patients who received glucocorticoids, human immunoglobulin, high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not. Error bars, SEM.

Data information: In (A-E), data were not normally distributed, and correlation was tested by Spearman correlation. In (F), statistical analysis was performed by paired two-tailed Man-Whitney U test.
The role of interleukin-6 in monitoring severe case of coronavirus disease 2019 (2020)
Figure 4

Figure 4. Variation in IL-6 level and radiological findings in severe COVID-19 patients during disease course.

IL-6 levels before and after treatment in three patient groups classified according to baseline IL-6 levels of ≥ 40 pg/mL (n = 11), ≥ 20 pg/mL (n = 6) or < 20 pg/mL (n = 9) (left). Two representative chest computed tomography (CT) scans before and after treatment from each group (right).

IL-6 levels at diagnosis and after disease progression in three exacerbated patients (left). Progressed radiological findings were recorded in two patients (right), while radiological assessment after treatment was not performed in the third patient due to poor general condition.

The baseline IL-6 level was 197.39 pg/mL in a 69-year-old female patient who showed high fever and dyspnea. IL-6 decreased to 9.47 pg/mL after treatment (day 8), while the symptoms were not relieved. The C-reactive protein (CRP) rebounded and procalcitonin (PCT) increased together with disease exacerbation. Follow-up chest computed tomography (CT) assessment was not performed due to poor general condition, whereas chest X-ray showed aggravated pneumonia. Follow-up sputum culture confirmed the exacerbation was caused by bacterial infection.

Data information: In (A), statistical analysis was performed by paired two-tailed Mann-Whitney U test. Blue dotted lines denote normal value or normal range.
The role of interleukin-6 in monitoring severe case of coronavirus disease 2019 (2020)
Figure 5

Figure 5. A case of a 59-year-old male patient diagnosed as severe COVID-19 on the fifth day from the onset of fever.

A) The chest computed tomography (CT) was normal five days after symptom onset. Disease aggravation was evidenced by progressed chest CT images ten days after symptom onset displaying bilateral multiple patchy ground glass opacities. Disease alleviation was evidenced by improved chest CT images 25 days after symptom onset.

B) Procalcitonin (PCT) levels stayed within normal range throughout the course of disease.

C) Lymphocyte count was normal five days after symptom onset, while firstly decreasing then returning to normal range during the course of disease.

D-E) Both abnormal C-reactive protein (CRP) (D) and IL-6 (E) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, both CRP and IL-6 further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.

F) The body temperature was elevated when CRP and IL-6 levels were rising, but returned to normal when CRP and IL-6. Levels were declining.

Data information: Blue dotted lines denote normal value or normal range.
Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy (2020)
Figure 1

Figure 1: TCHP interacts directly with PCM1

A, B (A) Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP, and FLAG-TCHP at N- and C- terminal. (B) Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP-FLAG C-terminal or constructs with deletions of the coiled-coil domain 1 (TCHP ∆1) and 2 (TCHP ∆2), as indicated in the scheme. For A and B: total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.

C, Anti-PCM1 immunoprecipitation from HUVECs cells and TCHP and PCM1 immunoblot.

D, Co-localization of TCHP-V5 and centriolar satellite proteins. HUVECs were transduced with TCHP-V5 lentivirus and stained for anti-PCM1, anti-CEP290, anti-CEP72 and anti-V5 antibodies. Scale bars, 25 µm and 5 µm in the inset. Right panel: the two-channel intensity correlation of pixels corresponding to regions identified with TCHP-V5 and satellite markers (n=90 cells; Pearson colocalization coefficient).

E, TCHP knock-down or control cells were stained for anti-PCM1 antibody. Panel below: quantification (n=50 cells; unpaired t-test; **p=0.004 vs control). Scale bars, 50 µm and 5 µm in the inset.

Data information: statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD.
Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy (2020)
Figure 2

Figure 2: TCHP regulates PCM1 and GABARAP stability

A, B (A) Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. PCM1 levels were analysed in the insoluble fraction. Laminin has been used as a loading control. Below panel: Quantification of PCM1 degradation. (one-way ANOVA; *p=0.0180 vs control time 0). (B) Condition as in (A), Western blot for anti-GABARAP and anti-ACTIN antibodies. Below panel: Quantification of GABARAP degradation. (one-way ANOVA; *p =0.0215 vs control time 0).

C, D (C) Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-PCM1 and anti-V5 antibodies. Laminin has been used as a loading control. (D) Condition as in (C), Western blot was probed for anti-V5, anti-GABARAP and anti-ACTIN antibodies. Below panels: Quantification of C (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0003 vs control CHX) and D (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0056 vs control CHX).

Data information: statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD.
Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy (2020)
Figure 3

Figure 3: Analysis of autophagy in TCHP-depleted endothelial cells

A, Immunofluorescent staining for LC3 and p62 in TCHP knock-down and control cells. Scale bars, 25μm. Lower panel: quantification. (n=90 cells, unpaired t-test; LC3: **p<0.0001 vs control; p62: **p<0.0001 vs control).

B, Western blot of p62 and LC3 during under normal culture condition or starved condition (HBSS) or the presence of BafA1 (2 hours) in TCHP knock-down or control cells. Lower panels: p62 quantification. (one-way ANOVA; **p=0.0003 vs control, ##p=0.0093 vs shTCHP); LC3 quantification. (one-way ANOVA; **p=0.0008 vs control, ##p=0.0011 vs shTCHP).

C, HUVECs were transduced with the tandem mCherry-EGFP-LC3 and with shRNA TCHP or control vectors. Left panels: Representative picture of mCherrry-EGFP-LC3 reporters. Scale bars, 25μm. Right panel: Quantification of the number of mCherry-only (red bars, autolysosomes) or double-positive (mCherry+/EGFP+; yellow bars, autophagosomes). (n=80 cells, one-way ANOVA; *p=0.0447 vs control; ##p=0.0007 vs shTCHP).

D, Ratiometric flow cytometric analysis of mCherrry-EGFP-LC3 reporters as in C. (one-way ANOVA; *p=0.0247 vs control; ##p=0.0088 vs shTCHP).

E, Quantification of long-lived protein degradation assay in TCHP knock-down and control cells by flow cytometry (one-way ANOVA; **p=0.0018 vs control, ##p<0.0001 vs shTCHP).

F, HUVECs were transduced with TCHP-V5 and control vectors. Western blot for anti-V5 and anti-p62 antibody during under normal culture condition or HBSS or BafA1 or Torin-1 treatment. Lower panel: quantification. (one-way ANOVA; **p=0.0044; #p=0.0117 vs TCHP-V5).

Data information: statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD.