EMBO/soda-nel huggingface dataset:
Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever (2022)
Figure 5

Figure 5: Exogenous ITA restores intracellular levels in ACOD1-deficient macrophages and restricts C. burnetii replication.

(A) GC-MS analysis of TCA cycle metabolites and ITA in BMM infected with NMII (MOI 10). BMM were infected for 4 hours, followed by a washing step. Where indicated, exogenous ITA was added at 2.5 mM ITA, 4-OI at 250 µM, after removing extracellular bacteria. Macrophages were harvested after 24 hours. Data shown are from two independent experiments (n=4 biological replicates).

(B) ITA, but not 4-OI or DMI, reduce NMII replication to WT levels in ACOD1-deficient BMM. Shown are qPCR data as C. burnetii GE normalized per cell (each dot represents one mouse and data were pooled together from 4 individual experiments with 2.5 mM ITA (n=8-9 mice per genotype) and two experiments with 1 mM ITA, 250 µM 4-OI and 250 µM DMI (n= 4 mice per genotype). Each dot represents one mouse. Bars show mean plus SD. * p<0.05, ** p<0.01 in Kruskal-Wallis test with Dunn's multiple comparisons test.

(C) BMM were infected with tdTomato-expressing NMII and analyzed by flow cytometry 96 hours after infection. ITA was added at 2.5 mM. Depicted is the percentage of BMM highly positive for tdTomato (as in Fig. 2D). Pooled data from two independent experiments, each dot represents mean values of duplicates from BMM of one mouse.

(D) NMII replication in human MDM. Infection was performed with MOI 10 for 4 hours followed by washing. Cells were lysed then directly or after 144 hours. Average values of replicate wells from 6 individual donors.

(E) Addition of ITA inhibits NMII replication in human macrophages. ITA at the indicated concentrations was added to MDM after infection with NMII MOI 10. Bacterial burden was determined after 144 hours as GE per cell by qPCR. Data were normalized to MDM infected but not treated with ITA (100 %). Average values of replicates from n=5-7 donors, error bars indicate SD. p-values (Wilcoxon matched-pairs signed rank test) are indicated.

(F, G) Treatment of mice with 1 mg of ITA i.p. on days 1, 3, and 5 after i.p. infection with NMII lowers bacterial burden in the organs on day 7. C. burnetii GE quantified by qPCR (F), representative images of liver sections and quantification of immunohistochemistry for C. burnetii in liver sections as in Fig. 3E (G). n=6-11 mice (for NMII infections), pooled from two experiments. Each dot represents one mouse. Bars show mean with SD. Asterisks indicate significance (Mann-Whitney test comparing treatment and control group of NMII infected Acod1-/- mice in specified organs, * p<0.05, ** p<0.01).
The ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex (2019)
Figure 1

Figure 1 - PC1 and PC2 express in Xenopus oocytes but yield no channel current.

A. Transmembrane topology of PC1 and PC2 proteins. The two proteins associate at the C-terminus through the coiled-coil domains and the extracellular side via the TOP domains. The GAIN domain and the GPS site in PC1 and the EF-hand motif in PC2 are indicated. The last six transmembrane domains of PC1 (shown in orange) share sequence similarity with PC2.

B. Western blot of oocyte lysate (left) and biotinylation-purified surface (right) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1/PC2 complex compared to either protein expressed separately. Anti-PC1 C-terminus antibody [29] recognized both full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1. A higher-glycosylated 130 kDa PC2 (star) band was only seen when PC1 is coexpressed.

C. Co-IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes. IP was done with an anti-FLAG antibody. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1 are indicated. Both 120 and 130 kDa bands of PC2 were seen in the IPed product.

D. Representative current-voltage relationship (I-V) curves (left) and a scatter plot and bar graph (right) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording. The current of the GOF PC2_F604P is included as a control. Currents at +60 mV are shown in the bar graph. Each point represents the recorded current from one oocyte. Oocyte numbers for scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD (***P < 0.001, Student's t-test).
The ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex (2019)
Figure 2

Figure 2 - PC2_L677A/N681A (AA) mutant is a new GOF channel.

A. Cryo-EM structure of PC2. Left: bottom view of PC2 homomeric tetramer showing the pore-lining S5-S6 from each subunit assemble into the channel pore. Right: side view of S5-S6 from two subunits showing the L677 and N681 (in red) contribute to the restriction at the lower gate. F604P (in cyan) on S5 and the three residues (in green) forming the selectivity filter are also indicated. The structure shown here is previously reported with Protein Data Bank (PDB) code 5T4D [20]. A grey dashed line indicates the path of the ion flow.

B. Representative I-V curves (left) and a scatter plot and bar graph (right) showing that the PC2_AA is a GOF mutant and gave rise to a larger current than PC2_F604P. Scatter plot and bar graph shows the average current sizes at +60 mV. The cations included in the bath solution, 100 mM Na+ and 2 mM Ca2+ in this case, are indicated by the thick-lined boxes here and in all the following figures. Oocyte numbers for bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).

C. Representative currents of PC2_F604P and PC2_AA mutants in the divalent ion-free bath solution, which contains 100 mM Na+.

D. Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2+.

E. Calcium-activated chloride channel (CaCC) blocker MONNA (10 μM) or niflumic acid (NFA) (1 mM) partially blocked the current recorded from the PC2_AA-injected oocytes in the 70 mM Ca2+ bath solution.
The ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex (2019)
Figure 3

Figure 3 - The PC1/PC2_AA complex channel has greater Ca2+ permeability than the homomeric PC2_AA channel.

A, B. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 100 mM Na+ (A), and 100 mM Na+ and 2 mM Ca2+ (B). Scatter plot and bar graphs show currents at +80 mV and -80 (or -70) mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, ***P < 0.001, Student's t-test).

C. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 100 mM Na+ and 2 mM Ca2+ solution in the absence or presence of 10 μM MONNA.

D. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 70 mM Ca2+. Scatter plot and bar graphs show currents at +80 mV and -70 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).

E. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 70 mM Ca2+ solution in the absence or presence of 10 μM MONNA.

F. Representative currents of PC2_AA (left) and PC1/PC2_AA (right) in solutions containing the indicated Ca2+ concentrations. All solutions contain 20 μM of MONNA to block CaCC current. Corresponding concentrations of NMDG+ were added to compensate the osmolarity. See Fig EV2 for currents of the full voltage scale and the details of solutions used.

G. Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1/PC2_AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone. For oocytes injected with PC2 alone, five times more concentrated PC2 RNA was injected to increase its surface expression. The purple dashed line indicates the background 45Ca uptake set by the measurement with the water-injected oocytes. Data were averaged from three independent experiments. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).

H. Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments.
The ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex (2019)
Figure 4

Figure 4 - Both the CTF and the TRP-Like domain (TLD) are sufficient for ion channel function of PC1 in the PC1/PC2 complex.

A. Transmembrane topology of PC1 and PC2 proteins, showing the NTF, CTF, and the TLD of PC1. The six transmembrane domains in TLD (indicated with the dashed line box) shares sequence homology with the transmembrane domains of PC2.

B, C, D. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ (B), 100 mM Na+ and 2 mM Ca2+ (C), or 70 mM Ca2+ (D). Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, *P < 0.05; ***P < 0.001 Student's t-test).

E. Scatter plot and bar graph showing that the current of PC1-CTF/PC2_AA is completely abolished when WT PC2 was used, or after introducing R4090W mutation in the putative pore region of PC1-CTF. Inserted are western blot images showing the expression of the corresponding proteins. Top: anti-PC1. Bottom: anti-PC2. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).

F, G, H. Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ and 2 mM Ca2+ (F), 100 mM Na+ (G), or 70 mM Ca2+ (H), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.

I. Co-IP followed by western blot showing the association between PC2_AA and the indicated full-length or fragments of PC1. Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.

J. Surface biotinylation followed by western blot showing the expression of the complexes formed between PC2_AA and full-length PC1, PC1-CTF, or PC1-TLD at the oocyte surface. Besides the blots shown in the figure, surface samples were also blotted with an anti-PC1 N-terminus antibody, and the cleaved NTF fragment was found associated to the plasma membrane in the full-length sample (shown in Appendix Fig S4). Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.
The ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex (2019)
Figure 5

Figure 5 - PC1-CTF/PC2_AA heteromeric channel has a different ion permeability to that of the PC2_AA homomeric channel.

A. Representative I-V curves of the PC2_AA and PC1-CTF/PC2_AA channels in divalent ion-free bath solutions containing 100 mM of indicated ions, showing the differences in reversal potential.

B. Bar graphs showing the markedly different reversal potentials of PC2_AA channel and the PC1-CTF/PC2_AA channel in bath solutions containing 100 mM of the indicated ions. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, ***P < 0.001, Student's t-test).

C, D. Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (C) and NMDG+ (Px: PNMDG) (D). These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.
The ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex (2019)
Figure 6

Figure 6 - Pore mutations in PC1 or PC2 led to changes in ion permeability of the PC1-CTF/PC2_AA channel.

A. TOP: a bottom view of a previously reported cryo-EM structure of PC1/PC2 channel (PDB code 6A70) [42], which is missing the NTF of PC1 and the intracellular C-terminal tails from both proteins. S10-S11 of one PC1 subunit and S5-S6 from three PC2 subunits assembled to form the pore. Bottom: the mutated amino acids in this experiment (in purple) are mapped on S10-S11 of one PC1 and S5-S6 of one PC2 subunit in the complex. The dashed grey line indicates the putative ion flow path. Since human PC1 was used for structure determination, mouse amino acids mutated in this study are indicated in parentheses. Due to the low resolution of the PC1 pore region in the structure, side chains are not seen for the three mutated amino acids at the putative selectivity filter region. E4068 (E4078 in human) is not solved in the structure, and it is labeled to indicate its approximate location in the structure.

B, C. Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions (B), indicating that these amino acids are essential for ion permeability. Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others (C), indicating a less important role of these amino acids in ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Representative I-V curves are shown in Fig EV5. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).

D, E. Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (D) and NMDG+ (Px: PNMDG) (E). Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.
The ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex (2019)
Figure 7

Figure 7 - GPS cleavage of PC1 is not necessary for channel activity of the PC1/PC2_AA channel.

A. Transmembrane topology of PC1 and PC2 proteins, showing the cleavage at the N-terminal GPS site (in circles), the "HLT" tripeptide where the cleavage happens, and the two mutations, L3040H and T3041V, that abolish GPS cleavage.

B, C. Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (B) or 70 mM Ca2+ (C). Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).

D. Surface biotinylation followed by western blot showing the surface expression of the PC1_T3041V/PC2_AA complex, but not the PC1_L3040H/PC2_AA complex, in Xenopus oocytes. PC2_AA is completely absent from the surface sample when PC1_L3040H was coexpressed, indicating that, in our experimental condition, almost all PC2_AA were in the complex with PC1_L3040H and trapped in the process of cell surface trafficking. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1, and 130 kDa PC2 (star) are indicated.
PHF6 promotes non-homologous end joining and G2 checkpoint recovery (2019)
Figure 1

Figure 1. RNA interference-based screen for G2 checkpoint recovery.

A) Experimental setup used to screen for chromatin regulators involved in the IR-induced G2 checkpoint response and recovery. Unless stated otherwise, 5 Gy of IR was used.

B) Results of three independent microscopy-based screens in U2OS cells. Knockdown of Luciferase (blue) and the positive controls PPM1D and βTrCP (red) are indicated. A low/high Z-score indicates less/more phospho-histone H3 positive cells compared to the control, reflecting reduced/increased numbers of cells going into mitosis in the respective knockdowns after 5 Gy of IR.

C) Deconvolution of the 41 primary hits in U2OS cells by four separate siRNA oligonucleotides. Grey represents a recovery value that is similar to the Luciferase control (<0.5 SD) and the red represents an altered recovery value (>0.5 SD) in two independent experiments.

D, E) U2OS cells (D) and RPE1 hTert immortalized primary epithelial cells (E) were transfected with siRNA oligos targeting the hits in a similar setup as described in A), but now analyzed for mitotic entry by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.

F) Graphical explanation of the known interactions between the identified hits through STRING pathway analysis.
PHF6 promotes non-homologous end joining and G2 checkpoint recovery (2019)
Figure 2

Figure 2. Classification of the screen hits.

A) Overview of the cellular mechanisms that influence DNA damage-induced G2 checkpoint recovery.

B) RPE1 cells were depleted for p53 in combination with the indicated proteins by siRNA, in conditions used for the primary screen (2 Gy of IR). Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.

C) U2OS cells were transfected with the indicated siRNA oligonucleotides in a setup similar to the primary screen, but with 10 Gy of IR and in the presence of ATM and ATR inhibitors. Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.

D, E) U2OS cells were transfected with the indicated siRNA oligos using the protocol of the primary screen and fixed for immunofluorescence after 0, 2 and 24 h after 5 Gy IR. Samples were stained with antibodies for γH2AX (D) or 53BP1 (E) and analyzed through automated confocal microscopy and specifically developed image analysis software. From one experiment two to four separate images with a total of at least 100 cells were analyzed for each knockdown. Shown are the number of γH2AX (D) and 53BP1 (E) foci per cell. In the box plots, the median with the first and third quartile is indicated, and the whiskers are drawn down to the 10th percentile and up to the 90th.
PHF6 promotes non-homologous end joining and G2 checkpoint recovery (2019)
Figure 3

Figure 3. PHF6 regulates G2 checkpoint recovery.

A) Graphical explanation of the effect of PHF6 on checkpoint recovery through DNA repair and p53.

B, C) U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 and subsequent recovery after 5 Gy of IR and the addition of nocodazole was analyzed by flow cytometry with MPM2 staining (B), or cells were lysed and analyzed using western blotting with the indicated antibodies (C). PPM1D and βTrCP were used as positive controls. Error bars represent the SEM of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (**p < 0.01, ***p < 0.001).

D, E) U2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 and recovery was determined after 2 Gy of IR using flow cytometry with pHH3 staining (D) or cells were lysed and analyzed using western blotting with the indicated antibodies (E). Error bars represent the SEM of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (**p < 0.01). The remaining PHF6 signal in the PHF6 KO sample (E) is likely due to a cross reaction at similar height as PHF6 (also see EV Figure 3C).

F, G) U2OS WT and PHF6 knockout cells were irradiated using 3 Gy of IR and cells were fixed at different time points for immunofluorescence. In (F) is presented 53BP1 IRIF intensity. In (G) is shown the number of γH2AX foci per cell. Error bars represent the SD of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (****p < 0.0001).

H) U2OS WT and PHF6 knockout cells were left untreated or irradiated with 3 Gy and processed after 1 h for comet assay analysis. The comet tail moment was analyzed in at least 50 individual cells. Error bars represent the SD of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (*p < 0.05, ****p < 0.0001).
PHF6 promotes non-homologous end joining and G2 checkpoint recovery (2019)
Figure 4

Figure 4. PHF6 affects recovery by controlling DNA repair via classical NHEJ.

A, B) U2OS cells stably expressing GFP-PHF6 and mCherry-NBS1 were laser-irradiated and cells were analyzed by time-lapse imaging. In (B), the PARP inhibitor Olaparib was added 30 min before laser-induced damage. At least 60 cells were analyzed in three individual experiments and error bars represent the SD (lower panel). Scale bar is 5 μM.

C) GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later the cells were fixed and analyzed by flow cytometry. Represented is the relative repair efficiency as compared to the Luciferase control. Error bars represent the SEM of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (**p < 0.01, ***p < 0.001).

D) Clonogenic survival assays of U2OS WT and PHF6 knockout cells that were depleted for luciferase or XRCC4 and treated with 2, 3 or 4 Gy IR. Shown is the relative survival as compared to the undamaged control. Error bars represent the SEM of three individual experiments.

E) GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later genomic DNA was extracted and repair of I-SceI cut-sites analyzed through junction analysis (n= independently derived sequences, see materials and methods for details).

F) Model showing early recruitment of PHF6 to DNA DSBs in a PARP1/2-dependent manner, possibly to remodel the chromatin for efficient DNA repair through classical NHEJ and thereby to promote recovery from the G2 checkpoint arrest.