EMBO/soda-nel huggingface dataset:
A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy (2013)
Figure 6

p62/SQSTM1 targets L. pneumophila‐containing vacuoles to the autophagosomes for degradation in WT murine macrophages. (A) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria. P62 is stained red with p62 antibody. White arrows show L. pneumophila colocalization with p62. (B) Percent colocalization of L. pneumophila with p62. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm. (C) BMDMs were infected with JR32 or the legA9 mutant for 6 h and the level of p62 protein was quantified in cell lysates by ELISA (performed in triplicate and the SD are too small to detect). BMDMs were nucleofected with scrambled control (sictrl) or siRNA against p62 (sip 62). Twenty‐four hours after nucleofection, murine macrophages were infected with JR32 or legA9 mutant with an MOI of 0.5 for 1 h (D and F) or 24 h (E and G). (H) Western blot analysis showing the downregulation of p62 at 24 and 48 h postnucleofection. (A–B and D–H) Data are shown as mean + SD of n = 3 and are representative of three independent experiments. (C) Data are representative of one experiment n = 3. Asterisks indicate significant differences (*p < 0.05, **p < 0.01; two‐tailed t‐test).
A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy (2013)
Figure 7

The permissiveness of BMDMs to legA9 mutant bacteria is independent of caspase‐1 or caspase‐7 activation. (A) Caspase‐1 KO (casp‐1−/−) macrophages were infected with L. pneumophila JR32 or legA9 with an MOI of 0.5 for 1, 24, 48, and 72 h. (B) CFUs were measured at the indicated time points. Levels of IL‐1β were detected in supernatants of WT or casp‐1−/− BMDMs infected with JR32 or the legA9 mutant after 24 h. WT BMDMs were either not treated (NT) or infected with L. pneumophila JR32 or legA9 mutant bacteria for 2 h. Activation of casp‐1 or casp‐7 was detected in cell extracts using (C) casp‐1 or (D) casp‐7 antibodies, respectively. (A–D) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.
A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy (2013)
Figure 8

Replication of the legA9 mutant is not due to extended macrophage survival. (A) BMDMs were not infected (NT) or infected with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5, 1 or 5 for 24 h. Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic (apoptosis) histone‐associated‐DNA fragments (ELISA). (B) BMDMs were either not treated (NT) or treated with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5 or 5 for 24 h. The fold change in LDH release was measured from the overall population of macrophages. (A–B) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.
Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration. (2015)
Fig 1

HIV induces autophagy in human macrophages.

(A) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (B) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h in the presence of pepstatin A, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (C) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h. Cells were then harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.
Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration. (2015)
Fig 2

HIV induces autophagy in human macrophages through TLR8.

(A) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (B) Macrophages transduced with shNS or shTLR8 from (A) were exposed to infectious HIV (+), AT-2-inactivated HIV (AT), or RNase/DNase I treated AT-2-inactivated HIV (R) or mock infected (-) for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; blue) and antibody to LC3B (green), SQSTM1 (magenta), HIV p24 and p17 (red). Co-localization of HIV and LC3B is represented by orange pixels, and co-localization of HIV, SQSTM1, and LC3B is represented by cream pixels. Scale bars indicate 10 μm. (D) Macrophages were transduced with shNS or ATG13 shRNA (shATG13) and analyzed for ATG13 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (E) Macrophages transduced with shNS or shATG13 from (D) were exposed to infectious HIV, 5 μg/mL ssRNA40, 5 μg/mL ssRNA41, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages were pretreated with 100 nmol/L bafilomycin A1 then exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV, LyoVec, 5 μg/mL ssRNA41, 5 μg/mL ssRNA40, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B, CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.
Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration. (2015)
Fig 3

Autophagy is decreased at later time points post-infection in human macrophages.

Macrophages were infected with HIV and cells harvested at 1, 3, 5, 7, and 10 days post-infection. (A) Cells were analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6. (B) Cells were harvested then subjected to flow cytometry analysis of saponin-resistant LC3B-II in macrophages. Representative histograms of cells displaying saponin-resistant LC3B-II are shown. (C) Cells were harvested, lysed then analyzed for CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6.
Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration. (2015)
Fig 4

HIV induces TFEB nuclear localization in human macrophages through TLR8.

(A) Macrophages were exposed to increasing concentrations of purified HIV for 24 h, harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (B) Macrophages were infected with HIV and at 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6. (C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; green) and antibody to TFEB (red) at the indicated times post-infection. Co-localization of TFEB and DAPI staining is represented in the image by orange pixels. Scale bars indicate 20 μm. (D) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h, harvested, lysed fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (E) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages transduced with shNS or shTLR8 from (E) were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were then harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration. (2015)
Fig 5

HIV-mediated autophagy induction is dependent upon TFEB.

(A) Macrophages transduced with non-specific scrambled shRNA (shNS), or TFEB shRNA (shTFEB) were infected with HIV for 10 days. Cells were lysed and analyzed for TFEB and ACTB content by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (B) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed, and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (C) Macrophages transduced with non-specific cDNA (Control), or TFEB cDNA (TFEB) then exposed to HIV. Cells were lysed and analyzed for TFEB and ACTB content by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (D) Transduced cells from (C) were exposed to purified HIV for the indicated time (minutes) harvested, lysed, and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration. (2015)
Fig 6

HIV-mediated autophagy and lysosomal gene expression is dependent upon TFEB.

(A) qRT-PCR analysis of mRNA expression of autophagy (UVRAG and ATG9B) and lysosomal (MCOLN1) genes 24 h post-exposure to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV. Data are reported as mean ± s.e.m., n = 4. (B) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (C) Macrophages from (B) were exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were harvested and qRT-PCR for UVRAG, ATG9B, MCOLN1 was performed. Data were normalized to the shNS mock infected control for each gene. Bar charts are reported as mean ± s.e.m., n = 4.
Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration. (2015)
Fig 7

HIV-mediated autophagy induction and nuclear translocation of TFEB is dependent upon BECN1.

(A) Macrophages transduced with non-specific scrambled shRNA (shNS), or BECN1 shRNA (shBECN1) were infected with HIV for 10 days. Cells were lysed and analyzed for BECN1 and ACTB content by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (B) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (C) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration. (2015)
Fig 8

HIV-mediated autophagy induction and nuclear translocation of TFEB is inhibited by HIV Nef.

(A) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (B) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death (2020)
Figure 1

Figure 1. Inhibition of efflux pump MRP1 synergizes with APR-246

Growth suppression in cancer cells after 72h of treatment with APR-246 +/- MRP1 inhibitor MK-571 as shown by the WST-1 assay (n ≥ 3, n shown in Table S1). Cross in heatmap indicates no data available.

Growth suppression in human dermal fibroblasts (HDF) by WST-1 assay after 72h of with APR-246 +/- MK-571 (n = 3).

Mean IC50 values (µM) for APR-246 +/- 20 μM MK-571 of 21 cell lines. *p < 0.0001, Wilcoxon test. Each dot represents one cell line. Mean IC50 values and n shown in Table S1.

ZIP synergy scores of most synergistic area sub-grouped according to TP53 gene status for 21 cell lines. Each dot represents one cell line. Scores and n are shown in Table S1.

Sub-G1 DNA content of OVCAR-3 cells (R248Q) after APR-246 +/- MK-571 treatment as assessed by propidium iodide staining at 48h (n = 3).

OVCAR-3 cell confluency by IncuCyte® during 72h of treatment with APR-246 +/- MK-571 (n = 3).

Growth suppression in HCT116 WT (n = 4) and R248W cells (n = 3) after 72h treatment with APR-246 +/- MRP1 inhibitor Reversan, as shown by WST-1.

Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53.

Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with MRP1 siRNA (n ≥ 5 and n ≥ 2 respectively) after 48h APR-246 treatment. Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs. Data and n of individual siRNAs are shown in Fig S1J, data is also part of Fig 7B.

Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector (EV) and MRP1 expression vector.

Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with empty vector (EV) and MRP1 vector after 72h APR-246 treatment (n = 4). Data is also part of Fig S7E.

Data information: TP53 status is shown for each cell line. Data are represented as mean ± SEM. See also Figure EV1 and Appendix Figure S1.
A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death (2020)
Figure 2

Figure 2. MK-571 enhances the anti-tumor activity of APR-246 in vivo and ex vivo

Growth of Eso26 xenografts (R248W mutant TP53) in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. p values determined by one-way ANOVA with Tukey's correction at day 18, n is indicated in the figure.

Kaplan-Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. Mice that were culled at ethical endpoints prior to reaching a tumor volume of >1400mm3 are marked as censored observations. p values were determined by Log-rank (Mantell-Cox) test between different treatment groups. n is indicated in the figure.

Quantification of Ki67 immunohistochemistry staining of Eso26 xenograft tumors at an early timepoint (5 days) or post treatment / late timepoint (>22 days) after treatment initiation with APR-246 (50mg/kg) +/- MK-571 (50mg/kg). Dots indicate individual tumors.

Quantification of cleaved caspase 3 immunohistochemistry staining of Eso26 xenograft tumors in same tumors as shown in Fig. 2C. Dots indicate individual tumors.

Representative images of hematoxylin/eosin (H&E) staining and immunostaining of Ki67, cleaved caspase 3 and p53, post treatment / late timepoint (>22 days after treatment initiation) with APR-246 (50mg/kg) +/- MK-571 (50mg/kg).

Growth suppression determined by the ATP-based CTG assay in colorectal cancer patient-derived organoids (colo-PDO) after treatment with APR-246 +/- MK-571 (n = 3).

IC50 values (µM) APR-246 +/- 20 μM MK-571 in indicated PDOs. *p = 0.04, paired t test (n = 3 except colo-PDO2 and eso-PDO3 where n = 2).

Organoid area (in black, left y-axis) and PI intensity (in red, right y-axis) of colo-PDO1 and colo-PDO2 as determined by image analysis 72h after treatment with APR-246 +/- MK-571 (n = 3).

Highest synergy score according to the ZIP model based on growth suppression as shown by CTG assay (ATP-based) or image analysis (Area and PI) in eso-PDOs and colo-PDOs. Scores are shown on a log scale. Score above 0 indicates synergy. Each dot indicates n for individual PDOs.

Data information: TP53 status is indicated. Data are represented as mean ± SEM. See also Appendix Figure S2 and Table S2.
A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death (2020)
Figure 3

Figure 3. Inhibition of MRP1 efflux pump activity increases 14C-APR-246/MQ accumulation in cancer cells

Chemical structure of 14C -APR-246 and its active product MQ.

14C accumulation (cpm/mg x ml-1) in 11 cell lines after 24h 14C -APR-246 +/- MK-571 treatment (n ≥ 3). *p = 0.0006, paired t-test. 14C-accumulation and n of individual cell lines as shown in Table S3.

14C accumulation (cpm/mg x ml-1) in HCT116 (WT and R248W) cells after 24h +treatment with 14C -APR-246 +/- MK-571 or Reversan (n ≥ 3, each dots in figure).

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells (n ≥ 3 and ≥ 2) transfected with MRP1 siRNAs after 24h of 14C-APR-246 treatment. Indicated values represent four individual MRP1 siRNAs and two individual negative control (Ctrl) siRNAs, where each dot is an individual siRNA. Mean of 14C-accumulation after treatment and n with individual siRNA sequences is shown in Table S4. *p = 0.04 and for R248W p = 0.06, Wilcoxon matched pairs signed rank test.

Data information: Data are represented as mean ± SEM. See also Figure EV3 and Appendix Figure S3.
A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death (2020)
Figure 5

Figure 5. APR-246 sensitivity is dependent on the presence of mutant p53, cellular thiol status and drug accumulation

Mean IC50 (µM) values of APR-246 in 17 different cell lines (n ≥ 3) as shown by WST-1. *p = 0.03, Mann Whitney test. Mean IC50 (µM) values for APR-246 treatment and n for individual cell lines are also shown in Fig 1C and listed in Table S1.

Box-and-whisker plot of Pearson correlations between PRIMA-1 area-under-the-curve (AUC) and the levels of 225 different metabolites from the DepMap portal. Central band indicates median, boxes indicate 25th and 75th percentile, and whiskers show 2.5th and 97.5th percentile outlier metabolites. High GSH and GSSG (in red) correlate with low PRIMA-1 sensitivity.

Total intracellular glutathione (GSH+GSSG) as shown by glutathione reductase (GR) re-cycling assay in H1299 R175H (tet-off) (+/- doxycycline) and H1299 -/- cells 24h after seeding (n = 3). Indicated values are fold change compared to mutant TP53 cells.

Total intracellular glutathione (GSH+GSSG) as shown by GR re-cycling assay in untreated HCT116 (R248W, WT and -/-) cells 48h after seeding (n = 3, 5 or 2 respectively). Indicated values are fold change to mutant TP53 cells. Part of this data is shown in Fig. EV4.

Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells (n = 2)

Correlation between 14C accumulation (cpm/mg x ml-1) after treatment with 25 µM 14C-APR-246 and IC50 (µM) values for APR-246 in 11 cell lines (n ≥ 3) (r = -0.66, p = 0.03, Pearson correlation). Each dot represents one cell line, 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3.

ABCC1 mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) in lung cancer (luad & lusc studies, *p<0.0001) and colon adenocarcinoma (coad study, *p=0.005) grouped into having no alterations or putative driver missense mutations in TP53. Statistical analysis by Mann Whitney test, n is indicated in the figure.

14C accumulation (cpm/mg x ml-1) in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells after 24h treatment with 14C-APR-246 (n = 2).

Growth suppression in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells treated for 72h with APR-246 as shown by WST-1 assay (n = 3).

Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.