EMBO/soda-nel huggingface dataset:
Growth-mediated negative feedback shapes quantitative antibiotic response (2022)
Figure 4

Fig. 4|Slower growth increases folA expression, irrespective of whether growth is reduced by trimethoprim or by nutrient limitation.
(A) Dependence of growth rate (black) and folA expression (green) on TMP concentration. Schematic: FolA expression was measured using a promoter-GFP reporter inserted at a neutral site in the genome (Materials and Methods).
(B) Growth rate (black) and folA expression (green) in the absence of TMP at different growth rates achieved by different ratios of αMG/glucose.
(C) Scatterplot of folA expression level with growth rate across all combinations of TMP concentrations and αMG/glucose ratios shown in A and B (Appendix Figure S9). Pearson's correlation coefficient ρ and p-value from two-sided t-test are shown. FolA expression is largely determined by growth rate, suggesting that this regulation is growth rate-dependent and not mediated by a specific molecular mechanism.
(D) Dependence of folA expression on TMP concentration at four different αMG/glucose ratios (0, 2.5, 5, and 10 as shown). Darker green indicates greater αMG/glucose ratio. FolA expression converges approximately to the same level at high TMP concentrations.
Data information: Black line in A shows Hill function fit as in Fig. 1A; other lines show polynomial fits of first (C) or second order (A,B) to guide the eye. Horizontal dotted green line shows folA expression level in the absence of TMP. Error bars show standard deviation of three biological replicates.
Growth-mediated negative feedback shapes quantitative antibiotic response (2022)
Figure 5

Fig. 5|Breaking the growth-mediated negative feedback loop steepens the trimethoprim dose-response curve.
(A) Schematic: FolA expression is controlled by varying the IPTG concentration and measured by flow cytometry using a GFP fusion to FolA. Shades of green indicate different FolA levels. Wells encircled in red indicate how the effect of inverted folA regulation (where folA expression decreases with increasing TMP concentration, starting from a high level) can be inferred; wells encircled in black illustrate the same for constant FolA expression.
(B) Growth rate as a function of TMP concentration for different paths through IPTG-TMP concentration space as illustrated in A (Materials and Methods). Constant FolA is shown in black and inverted FolA regulation in red. Wild-type dose-response curve (blue line; fit from Fig. 4A) is shown for comparison.
(C) Steepness of the dose response curve (quantified as dose-sensitivity n) for the three cases in B. Inset: Normalized FolA expression level as a function of TMP concentration for the three cases in B; WT (blue) shows fit from Fig. 4A; colors as in the bar chart and in B.
Data information: Error bars in B show standard deviation of the measured growth rates used for interpolating the values shown; the entire experiment was replicated once (Materials and Methods). Error bars in C show standard deviation of parameter estimates from Hill function fit.
Efficacy of systemically delivered temozolomide-activated phage-targeted gene therapy in human glioblastoma. (2019)
Figure 1

Figure 1- Targeted transduction of human glioblastoma cell lines and induction of RGD4C/AAVP-Grp78 by TMZ. A) Immunofluorescence staining of LN229, U87and SNB19 tumor cells with primary antibodies against αv, β3 or β5 integrin subunits. Scale bars, 20μm for LN229 and SNB19, and 50μm for U87. B) Targeted gene delivery into LN229, U87 and SNB19 cells by RGD4C/AAVP-Luc carrying the Luc reporter gene. Cells were seeded in 48 well plates before transduction. Non-targeted/AAVP-Luc was used as negative control for targeted transduction. Results represent the average Relative Luminescence Units (RLU)/1μg protein. Data shown are representative of two experiments, n=3. C) Western blot analysis of the endogenous Grp78 protein expression levels in LN229, U87 and SNB19 cells following treatment with increasing concentrations of TMZ. Grp78 level was normalized to GAPDH. Data shown in right panel are representative of two experiments, n=3. D) Induction of Grp78 promoter activity by TMZ in RGD4C/AAVP-Grp78-Luc cells. LN229, U87 and SNB19 cells stably transduced with RGD4C/AAVP-Grp78-Luc or RGD4C/AAVP-CMV-Luc were grown in the presence of TMZ for the indicated times. Results represent the average RLU/1μg protein. Data shown are representative of two experiments, n=3. Two-way ANOVA with Bonferroni correction (GraphPad Prism 6) was used for data analysis.

Data information: Data are expressed as mean + SEM.
Efficacy of systemically delivered temozolomide-activated phage-targeted gene therapy in human glioblastoma. (2019)
Figure 2

Figure 2- TMZ activation of the UPR pathway and subsequent enhancement of glioblastoma cell killing in vitro by the RGD4C/AAVP-Grp78-HSVtk and GCV. A) TMZ induction of phospho-eIF2α and ATF6-p90 expression. Human glioblastoma cells transduced with RGD4C/AAVP-Grp78 were analyzed by Western blot following treatment with TMZ (100 μM for LN229 and SNB19, 60 μM for U87). GAPDH was used as a control. B) RT-PCR analysis of constitutive expression and splicing of XBP1 in glioblastoma cells transduced with RGD4C/AAVP-Grp78, in the presence of TMZ. Sizes of the PCR products were 289 bp for unspliced XBP1 and 263 bp for spliced XBP1, the lower size band is not specific. Time points were selected among those tested in the Western blot in (A), subsequently the image containing 0, 1 and 2 hours was juxtaposed to images of 6, 12 and 24 hours. A white line has been added between the gel pieces that have been juxtaposed. C) Tumor cell killing in vitro by the HSVtk/GCV approach. Glioblastoma cells stably transduced with RGD4C/AAVP-Grp78-HSVtk or RGD4C/AAVP-CMV-HSVtk were treated with either GCV (10 μM) or TMZ (100 μM for LN229 and SNB19, 60 μM for U87) or combination of both GCV and TMZ. Cells were stained with MitoSOX and analyzed by FACS at day 4 post-treatment. Data shown are representative of three experiments, n=3. Two-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 6) was used for data analysis.

Data information: Data are expressed as mean + SEM.
Efficacy of systemically delivered temozolomide-activated phage-targeted gene therapy in human glioblastoma. (2019)
Figure 3

Figure 3- Systemic targeting of orthotopic glioblastoma with RGD4C/AAVP-Grp78-HSVtk.

A) Immuofluorescent staining for αv integrin expression in brain sections including intracranial tumor (T) and the surrounding healthy brain (B). Scale bar, 100μm. B) Co-staining of CD31 (red) and phage (green) in brain sections comprising tumor and healthy brain. Scale bar, 100μm. A high-magnification view from the low-magnification insert of the tumor section is shown. Scale bar, 25 μm. C) Quantification of the relative homing ability of RGD4C/AAVP-Grp78-HSVtk to brain tumors, brain tissue and pancreas after intravenous administration. The data were normalized both to non-targeted vector and to tissue weight then expressed as relative TU. Data shown are representative of two experiments, n=5. One-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 6) was used for data analysis. D) Quantification of the vector dose reaching orthotopic GBM in mice upon intravenous administration of 5x1010 TU of RGD4C/AAVP vector. The vector amount, in tumors, was expressed as TU/g tumor tissue. Data shown are representative of two experiments, n=5.

Data information: Data are expressed as mean + SEM.
Efficacy of systemically delivered temozolomide-activated phage-targeted gene therapy in human glioblastoma. (2019)
Figure 5

Figure 5- Histological analysis of intracranial U87-derived glioblastoma after therapy. A) Hematoxylin and eosin staining (H&E) and immunostaining for the proliferation marker protein Ki67 of representative tumor sections from all experimental groups, Scale bars, 200 μm for H&E and 100 μm for Ki67. B) Blood vessel staining with CD31 (red). DAPI staining of the sections is shown in blue. Scale bar, 200 μm. C) Tumor immunostaining for apoptosis using an anti- caspase-3 antibody. Scale bar, 100 μm.

T, tumor; B, brain. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings.
Efficacy of systemically delivered temozolomide-activated phage-targeted gene therapy in human glioblastoma. (2019)
Figure 6

Figure 6- Characterization of the human primary GBM, then analysis of targeted gene delivery. A) Fluorescence-activated cell sorting (FACS) analysis with antibodies against Sox-2, Nestin and CD133 stem cell markers. B) Immunofluorescent staining for αv, β3 or β5 integrins in primary HSJD-GBM-001 and G26 GBM cells. C) Targeted transduction of HSJD-GBM-001 and G26 cells, over a time course, by RGD4C/AAVP-Luc. Non-targeted/AAVP-Luc was used as negative control. Results represent RLU/1μg protein of triplicate wells. Data shown are representative of two experiments, n=3. Scale bar, 100 μm.

Data information: Data are expressed as mean + SEM.
Efficacy of systemically delivered temozolomide-activated phage-targeted gene therapy in human glioblastoma. (2019)
Figure 7

Figure 7- Integrin expression in human primary normal cells, then analysis of targeted gene delivery. A) Immunofluorescent staining for αv, β3 or β5 integrins in human normal astrocytes, chondrocytes, skin and lung fibroblasts. B) Treatment of chondrocytes, astrocytes, lung and skin fibroblasts with RGD4C/AAVP-Luc vector, alone or in the presence of TMZ. Non-targeted/AAVP-Luc was used as negative control. Results represent RLU/1μg protein. Data shown are representative of two experiments, n=3. Scale bars, 160 μm for chondrocytes and 80 μm for all other cells.

Data information: Data are expressed as mean + SEM.
Efficacy of systemically delivered temozolomide-activated phage-targeted gene therapy in human glioblastoma. (2019)
Figure 8

Figure 8- Targeted systemic gene therapy against orthotopic primary HSJD-GBM-001 with RGD4C/AAVP-Grp78-HSVtk plus GCV and combination with TMZ. A) BLI of luciferase of representative tumor-bearing mice from all experimental groups at day 19 post therapy initiation. B) Survival benefit for tumor-bearing mice from all treatment groups. Data shown are representative of two experiments, n=6. Statistical significance was determined by Kaplan-Meier method (Kaplan-Meier survival fractions). Log-rank (Mantel-Cox) test was used. C) H&E staining, Ki67 immunostaining, and evaluation of apoptosis in tumors by using an anti- caspase-3 antibody staining of representative tumor sections from all experimental groups. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings. Arrow points to apoptotic cells. D) Immunostaining of tumors with an antibody against CD133. Scale bars, 100 μm.
Efficacy of systemically delivered temozolomide-activated phage-targeted gene therapy in human glioblastoma. (2019)
Figure 9

Figure 9- RGD4C/AAVP-Grp78-HSVtk plus GCV gene therapy and combination with TMZ against orthotopic murine GL261 GBM. A) Immunostaining analysis for αv, β3 or β5 integrins in GL261 cells. Scale bar, 50 μm. B) BLI of Luc of representative tumor-bearing mice from all experimental groups at day 34 post treatment. C) Total tumor Luc intensities in each experimental group before treatment, on day 10 post cell implantation, and after therapy, on day 34 post cell implantation. Data shown are representative of two experiments, n=6. Data analysis was done by two-way ANOVA and Bonferroni's multiple comparison test. D) Survival benefit for tumor-bearing mice from all treatment groups. Data shown are representative of two experiments, n=6. Statistical significance was determined by Kaplan-Meier method (Kaplan-Meier survival fractions). Log-rank (Mantel-Cox) test was used. E) Weights of C57BL/6J mice bearing intracranial GL261 tumors, from all experimental groups, before and after treatment. Data shown are representative of two experiments, n=6.

Data information: Data are expressed as mean + SEM.

Expanded View Figure Legends
Potent neutralization of 2019 novel coronavirus by recombinant ACE2-Ig (2020)
Figure 2.

ACE2-Ig potently neutralizes viruses pseudotyped with S glycoproteins from the SARS CoV and 2019 nCoV.HIVs pseudotyped with the S glycoprotein from CoVs were incubated with different fusion proteins for 1 h before infection. Luciferase activities in target cells were measured, and the percent neutralization was calculated.
Potent neutralization of 2019 novel coronavirus by recombinant ACE2-Ig (2020)
Figure 3.

Inhibition of cell fusion by ACE2-Ig.Potent inhibition of cell fusion mediated by the SARS-CoV (left) or 2019 nCoV (right) spike (S) glycoprotein. Cells expressing diffferent S glycoprotein were incubated with indicated fusion proteins and mixed with ACE2-expressing cells. The activity of the reporter gene, β-gal, was measured as a correlate of fusion. The curves represent the best fit to the experimental data and were used for calculation of the IC50. Bars indicate the S.E.
Proline biosynthesis is a vent for TGFβ-induced mitochondrial redox stress (2020)
Figure 2

Figure 2: TGFβ promotes proline synthesis from glutamine for collagen production.

(a) Comparison of the amino acid representation in all human proteins and all collagens. The x-axis denotes individual amino acids shown by single letter code.

(b) Glycine and proline representation in protein acid hydrolysates of untreated NIH-3T3 cells and their secreted ECM, produced as in Figure 1d. Amino acids were measured by GC-MS.

(c-h) Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells were treated with TGFβ (2 ng/mL for all experiments) or mock for 48h, and the medium was replaced (including all treatments) with DMEM lacking L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolic steady state was reached at this time point (see kinetic labeling curves in Figure EV3b). Metabolites were measured by LC-MS. The left graph shows the total pool size normalized to the packed cell volume; the right side shows the percent labeling. αKG, alpha-ketoglutarate; AU, arbitrary units.

(i) Glutamine flux into glutamate, αKG and proline in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells.

(j) Abundance of indicated metabolites in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells. Metabolites were measured by GC-MS.

(k) Schematic of [U-13C] Gln tracing into proline in collagen I-α1 (CO1A1) in NIH-3T3 cell-derived ECM. Expected m/z of unlabeled (m+0) and fully labeled proline (m+5 for one proline, m+10 for two prolines) for the analytical peptide are shown.

(l) Representative MS2 spectra of the CO1A1 peptide in ECM generated with unlabeled ([12C] Gln, left) and fully labeled glutamine ([U-13C] Gln, right). Peaks representing fully labeled (m+5, m+10) proline are highlighted in red. Peptides were measured by LC-MS.

(m) Relative abundance of the analytical peptide containing unlabeled (m+0) or one/two fully labelled prolines (m+5/m+10) from the CO1A1 peptide in ECM derived from NIH-3T3 cells treated with TGFβ or mock in the presence of fully labelled L-glutamine ([U-13C] Gln). Peptides were measured by LC-MS. The amount of ECM analyzed was normalized to the packed cell volume of cells grown on a parallel plate under identical conditions.

Data information: P-values were calculated by two-sided, unpaired t-test with Welch's correction (b, j, m) or two-way ANOVA with Holm-Sidak's multiple comparison test (i). Bars represent the mean + SD. n=20381 (All proteins), n=44 (Collagens) (a); n=4 (Cells), n=3 (ECM) (b); n=3 (c-j, m). A representative experiment is shown (l).
Proline biosynthesis is a vent for TGFβ-induced mitochondrial redox stress (2020)
Figure 4

Figure 4: TGFβ-induced stimulation of mitochondrial bioenergetics is required for increased proline biosynthesis.

(a, b) Mitochondrial basal respiration (a) and FCCP-induced maximal respiration (b) of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or mock for 2h, 4h, 8h, 24h or 48h, based on Seahorse OCR measurements. The relative values were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point.

(c) Left: western blot of NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX. MitoTPNOX or mitoLbNOX expression is detected by probing for their FLAG-tag. Right: NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are normalized to mock-treated empty vector expressing cells.

(d) NIH-3T3 cells were treated with rotenone, antimycin, myxothiazol or vehicle control for 1h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.

(e) NIH-3T3 cells were treated with cobalt chloride (CoCl2) or vehicle control for 6h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.

(f) NIH-3T3 cells were treated with TGFβ or mock for 48h, and FCCP or vehicle control was added for the last 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.

(g) NIH-3T3 cells were treated with TGFβ or mock for 48h, and oligomycin or vehicle control was added for the last 1h or 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.

Data information: P-values were calculated by two-way ANOVA with Holm-Sidak's multiple comparison test (c, g), by one-way ANOVA with Holm-Sidak's multiple comparison test (d, f) or by two-sided, unpaired t-test with Welch's correction (e). Bars in (c-g) represent the mean + SD; data in (a, b) represent the mean +/- SD. n=5-6 (a, b); n=3 (c-g).
Proline biosynthesis is a vent for TGFβ-induced mitochondrial redox stress (2020)
Figure 5

Figure 5: Proline biosynthesis is a vent for TGFβ-induced mitochondrial redox stress.

(a) NIH-3T3 cells were treated with TGFβ or mock for 1h, 6h, 24h or 48h. ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment (Red line). Proline abundance was measured by GC-MS (Blue line). The relative levels were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point.

(b) Western blot of NIH-3T3 cells were treated with TGFβ or mock for 1h, 6h, 24h or 48h.

(c) NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are normalized to mock-treated sgCtrl expressing cells.

(d) Western blot of NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 and treated with TGFβ or mock for 6h in the presence of 0.5% FBS.

(e) NIH-3T3 cells expressing mito-Grx1-roGFP2 or cyto-Grx1-roGFP2 were treated with TGFβ or mock for 6h or with 100 µM H2O2 as a control. Emission was measured with a 520/10nm filter after excitation with 405nm and 488nm lasers using flow cytometry. Oxidized roGFP2 gains excitability at 405nm and loses excitability at 488nm. Oxidation status is expressed as percentage of maximal oxidation which was determined by treating cells with 5 mM H2O2 for 5 min before harvest.

(f) NIH-3T3 cells expressing sgCtrl or sgP5CS-2 were treated with TGFβ or mock for 48h in the presence of 0.15 mM proline. ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment.

Data information: P-values were calculated by two-way ANOVA with Holm-Sidak's multiple comparison test (c, f) or by one-way ANOVA with Holm-Sidak's multiple comparison test (e). Bars represent the mean + SD (c, e, f); data in (a) represent the mean +/- SD. A representative experiment is shown (b, d).
Biochemically validated structural model of the 15-subunit intraflagellar transport complex IFT-B (2022)
Figure 4

Figure 4: IFT81/74 has two separate binding sites on IFT88/70/52/46

(A) The blue dotted lines indicate the IFT46 crosslinks to the C-termini of IFT81 and IFT74. These crosslinks map to a 43 residue stretch from K575 to K618 in IFT81 and a 27 residue stretch in IFT74 bordered by K581 and K608. An additional crosslinking pair was identified between IFT46 and IFT27. (B) K411 and K415 of IFT52 crosslink to K598, K600 and K607 of IFT81. These crosslinks are labelled as in panel (A). (C-D) The C-terminal domains of IFT52 and IFT46 co-purify with the IFT81460-C/74460-C/27/251-136 protein complex (C) but not with a protein complex that is missing residues 623-C of IFT81 and 615-C of IFT74 (D). The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition. (E) IFT811-385/74128-390 crosslinking network with IFT88, IFT70 and IFT52 labelled on the AF predicted model of Chlamydomonas IFT88120-713/70/52/811-385/741-390. The N-terminal 128 amino acids of IFT74 are predicted to be unstructured and omitted from the figure for clarity. The box shows a zoom-in on the IFT81/74/88 interaction interface. (F) The predicted alignment error plot of the Chlamydomonas IFT88120-713/70/52/811-385/741-390 complex shown in panel (E). (G) Coomassie stained SDS PAGE gel of a GST-pulldown using the CrIFT88His-GST/70/521-430/46 complex immobilized on GST beads as bait and the CrIFT811-387-GFP/74135-420 complex used as prey. The band highlighted by an * corresponds to pulled down CrIFT74135-420. CrIFT811-387-GFP runs on top of IFT70 and is not visible in the pull-down.
Biochemically validated structural model of the 15-subunit intraflagellar transport complex IFT-B (2022)
Figure 6

Figure 6: Dissecting the interaction between IFT80, IFT172, IT57 and IFT38

(A) AphaFold predicted structure of IFT1721-785/80. (B) Pull-down experiments with purified IFT80Venus immobilized on GFP-binder beads and either IFT172 or IFT172968-C. IFT80 pulls down full length IFT172 but not the truncated version lacking the N-terminal 967 residues. (C) AlphaFold predicted structure of IFT80 in complex with IFT38 maps the interaction at the interface between the BP1 of IFT80 and the CH-domain of IFT38. (D) Comparison of the solution scattering curve of CrIFT80/38 as measured by SAXS and the calculated scattering curve for the IFT80/38 structural prediction. A ꭓ2 value of close to 1 indicate an excellent fit between measurement and calculation. (E) AlphaFold predicted structure of CrIFT172 and CrIFT57 shows interaction via the BP1 and CH domains. On the right panel, two residues in the CH-domain of IFT57, which are located at the interface with IFT172, are highlighted. (F) GST tagged CrIFT1721-968 pulls down the CH-domain of CrIFT571-234 but not the mutated CH-domain of CrIFT57 where E77 and K84 residues were replaced by alanines. (G) Superimposition of the structure of BP1 of CrIFT80 (colored in red ruby) in complex with the CH-domain of 38 (colored in beige) with the BP1/CH domains of CrIFT172/57 (colored in teal and hot pink, respectively) shows a conserved mechanism of interaction different from the canonical tubulin binding mode exhibited by CH domains.