EMBO/soda-nel huggingface dataset:
Voltage-independent GluN2A-type NMDA receptor Ca2+ signaling promotes audiogenic seizures, attentional and cognitive deficits in mice. (2021)
Fig. 8

Associative learning in GluN2A(N615S) expressing mice.

a Odor discrimination. Grin2aS/S learned to discriminate between amyl acetate and ethyl butyrate in a rewarded go/no go-paradigm (250 trials for each odor stimulus, pseudo-randomized, and counterbalanced by stimulus identity across animals). Acquisition was similar for all three genotypes. b Simple visuo-tactile discrimination. Grin2aS/S mutant mice showed normal acquisition in the simple discrimination T-maze task and were able to associate a specific visuo-tactile insert (black foam versus light blue toweling) with a milk reward. c Morris watermaze. In the standard Morris watermaze task (left graph) the path length to reach the hidden platform decreased across training blocks for Grin2a+/+ and Grin2a+/S mice but not for Grin2aS/S mice. Two probe trials were conducted after 12 and 24 training trials (P1 and P2, respectively) during which the platform was removed from the pool. At both P1 and P2 (right bar graphs) the Grin2aS/S mice failed to search for the platform in the target quadrant. AdjL adjacent left, Target fixed location of the hidden escape platform during acquisition, AdjR adjacent right, Opp opposite of target quadrant. Dashed lines indicate chance levels. d Rewarded alternation. (left) In the T-maze rewarded alternation task (right) spatial working memory performance was substantially impaired in Grin2aS/S mice. e Contiguous task. In the contiguous version of the conditional T-maze task (with floor inserts covering the entire T-maze; white Perspex versus gray wire mesh), Grin2a+/+ and Grin2aS/S mice were able to associate a particular floor insert with the location of the reward in either the left or the right goal arm. f Discontiguous task. Separate groups of mice were trained in the discontiguous version of this conditional task, in which the floor insert cues were now limited to the start arm only. Grin2a+/+ mice readily acquired the task, but Grin2aS/S mice failed to learn. The numbers of mice are shown in brackets or below the bars of bar graphs. Chance levels (Ch.) are indicated by dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 8).
A luminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions (2018)
Figure 1

Figure 1: The LuTHy procedure.

A Schematic representation of the workflow of the LuTHy method. Expression vectors encoding NL and PA-mCit-tagged hybrid proteins are cloned and co-transfected into mammalian cells. Binary interactions are detected with a double-readout; first, in vivo with in-cell bioluminescence resonance energy transfer (BRET) and second, ex vivo with a luminescence-based co-precipitation (LuC).

B Schematic representation of control proteins and fusion proteins of interest for investigating the interaction between PA-mCit-BAD and NL-BCL2L1 in proof-of-principle LuTHy experiments.

C Calculated BRET ratios for the indicated protein pairs. The positive control protein PA-mCit-NL and the interacting fusion proteins NL-BCL2L1 and PA-mCit-BAD show high BRET ratios.

D Calculated LuC ratios for the indicated protein pairs. The positive control proteins PA-NL and PA-mCit-NL and the interacting proteins NL-BCL2L1 and PA-mCit-BAD show high LuC ratios. Data are representative of more than three independent experiments. All values are mean ± sd. Significance was calculated by one-way ANOVA followed by Dunnett's multiple comparisons post-hoc test (n=3, DF=8, F=211.3); ***p<0.001.
A luminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions (2018)
Figure 4

Figure 4: Effects of missense mutations and small-molecules on PPI detection with LuTHy.

A Live-cell bioluminescence imaging (BLI) of HEK293 cells co-producing the indicated fusion proteins. mCitrine was excited at 500 nm, emitted fluorescence was detected at 535 nm. After substrate addition, short (460) and long (535) band-pass (BP) filters in a dual-view adapter were used to detect the emitted luminescence simultaneously at the respective wavelengths. BRET images were calculated by dividing the 535 BP by the 460 BP images using ImageJ. Scale bar = 20 μm.

B Quantification of the rapamycin-induced interaction between NL-FKBP12 and PA-mCit-FRB. Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin. After an additional incubation for 4 h BRET was quantified upon substrate addition. After cell lysis in buffer with indicated rapamycin concentrations, LuC ratios were determined. EC50 values were obtained with non-linear curve fitting.

C Effects of Nutlin-3 on the interaction between NL-MDM2 and PA-mCit-p53. Same procedure as in D) except for 6 h treatment with Nutlin-3. Data are representative of at least two independent experiments. All values are mean ± sd.

D Time resolved HSF1 oligomerization by Hsp90 inhibition. HEK293 cells co-producing NL-HSF1 and PA-mCit-HSF1 for 24 h were transferred to 384-well plates containing Ganetespib or Geldanamycin to reach a final concentration of 1 µM. Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control. Data are the mean of two biological replicates. All values are mean ± sem. Significance was calculated by two-way ANOVA followed by Dunnett's multiple comparison's post-hoc test.

E Schematic depiction of a heat shock experiment. HEK293 cells co-producing NL-HSF1/PA-mCit-HSF1 for 48h were subjected to a heat shock at 42°C for 6 h, after which the cells were recovered at 37°C for 24h. Luminescence was measured at the indicated time points: immediately before the heat shock, during the heat shock at 90, 180 and 360 min and after a recovery phase of 24 h. Calculated cBRET ratios were normalized to a control plate that was not heat treated. All values are mean ± sem from three independent experiments. Significance was calculated by one-way ANOVA followed by Dunnett's multiple comparisons post-hoc test; *p<0.05.
A luminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions (2018)
Figure 5

Figure 5: Systematic investigation of the impact of fusion-tag orientation

A Domain overview of VCP and 10 UBX-domain containing proteins. The UBX proteins bind with their C-terminally located UBX-domains to the N domain in VCP, with the exception of UBXD1 that binds to the C-terminus of VCP via a PUB domain.

B,C,D VCP and the UBX domain-containing proteins were co-expressed as N- and C-terminal NL and PA-mCit fusion proteins in eight different orientations for which cBRET (B) and cLuC (C) ratios were generated. cBRET and cLuC values over the threshold of ≥0.01 and ≥0.03, respectively, are colored in cyan.

D The cBRET and cLuC ratios were normalized to the highest of the obtained eight values to easily identify the orientation among the eight tested interactions with the highest cBRET and cLuC value.
STAMP2 increases oxidative stress and is critical for prostate cancer. (2015)
Figure 1

STAMP2 expression during PCa progression

A Immunohistochemistry was used to assess STAMP2 expression in normal and malignant human prostate specimens. Tissue microarrays (TMAs) with normal prostate (n = 27) and early-stage (Gleason grade < 7) (n = 35) or late-stage prostate tumors (Gleason grade ≥ 7) (n = 149) were subjected to immunohistochemistry as described in Materials and Methods. Representative images are shown.

B Quantification of cytosolic STAMP2 staining in TMAs is shown in (A). One-way analysis of variance (ANOVA) with a post hoc test, *P < 0.0001. Error bars indicate SEM.

C, D The details of cytosolic (C) and membrane (D) STAMP2 staining in the TMAs described in (A).
STAMP2 increases oxidative stress and is critical for prostate cancer. (2015)
Figure 2

STAMP2 promotes PCa growth in vitro and in vivo

A LNCaP cells were transfected with either control siRNA or two independent STAMP2 siRNAs, and membrane fractions of the cells were prepared followed by Western blotting analysis. STAMP1 is used as a loading control.

B LNCaP cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h before being transfected with the indicated siRNAs. The cells were then cultured for the indicated times, and cell growth was measured by Cell Counting Kit-8. n = 3, *P < 0.0001. Error bars indicate SD.

C LNCaP cells were cultured and transfected with the indicated siRNAs as described in (B). After transfection, the cells were cultured for 10 days. The colonies formed were stained and photographed.

D LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were treated with or without 1 nM R1881. The cells were then collected, and membrane fractions were prepared followed by Western blot analysis.

E LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured for the indicated times, and the cell numbers were determined using a hemocytometer. n = 3, *P = 0.047; **P = 0.0046. Error bars indicate SD.

F LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 weeks as described in Materials and Methods. The plates were then stained and photographed.

G Quantification of data from (F). n = 3, *P = 0.022. Error bars indicate SD.

H LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were subcutaneously implanted into male SCID mice. Tumor size was measured after 8 weeks. n = 9, *P = 0.037. Error bars indicate SEM.

I VCaP cells were transfected with the indicated siRNA as described in (B). After transfection, VCaP cells were cultured for 2 weeks. The colonies formed were stained and photographed.

J Quantification of the data from (I). n = 3, *P < 0.0001. Error bars indicate SD.

Data information: In (B, E, G, H, and J), Student's t-test was performed to analyze the statistical significance. Source data are available online for this figure.
STAMP2 increases oxidative stress and is critical for prostate cancer. (2015)
Figure 3

STAMP2 affects cell cycle progression and increases sensitivity of PCa cells to apoptosis-inducing drugs

A LNCaP cells were transfected with either control or STAMP2-specific siRNA. Two days after transfection, cells were subjected to cell cycle analysis as described in Materials and Methods. Representative histograms showing PI-stained cells are shown.

B The proportion of cells in each stage of the cell cycle from the experiment in (A) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.0002; **P = 0.0056; ***P = 0.0099. Error bars indicate SD.

C Two days after transfection with the indicated siRNAs, LNCaP cells were harvested and cell lysates were prepared and subjected to Western blot analysis with the indicated antisera. Representative blots of three independent experiments are shown.

D LNCaP cells were transfected with either control or STAMP2-specific siRNA. Three days after transfection, cells were treated with either 50 ng/ml TRAIL or 20 μmol/l LY294002 (LY) for 24 h, or both agents for 6 h, and then subjected to TUNEL/FACS analysis. Representative histograms of TUNEL-stained cells are shown. FL1-H refers to the gating of the cells for the TUNEL staining with fluorescence measurement.

E The extent of apoptosis from the experiment in (D) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.027; **P = 0.0058; ***P = 0.0016. Error bars indicate SD.

F LNCaP cells were transfected with either control or two independent STAMP2-specific siRNAs, ST2-1 and ST2-2. Three days after transfection, cells were treated in the same way as in (D). Whole-cell lysates were prepared and subjected to Western blot analysis with the indicated antisera.

G LNCaP cells stably expressing either control or STAMP2 vector were treated in the same way as in (D). Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies.

Source data are available online for this figure.
STAMP2 increases oxidative stress and is critical for prostate cancer. (2015)
Figure 4

STAMP2 expression is associated with development of castration resistance of PCa

A STAMP2 expression was determined by IHC of a neoadjuvant hormone therapy (NHT) TMA containing samples from hormone naïve (untreated) (n = 28), NHT 1–6 months (n = 31), NHT 7–12 months (n = 41), and patients with PSA recurrence (n = 21) as indicated. Representative images are presented.

B Quantitative presentation of the data from (A). Unt, untreated; Recur, PSA recurrence. The Kruskal–Wallis test was used for significance analysis. *P = 0.0499. Error bars indicate SEM.

CBoxplots of STAMP2 expression levels in normal prostate tissue, primary prostate cancer, and metastatic CRPC were analyzed in two independent GEO datasets (GSE6919 and GSE35988). The values of individual samples are presented as dots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles. The statistical significance, indicated in the tables at the bottom, was determined by Student's t-test.

D CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. mRNA was extracted from the tumors and used for qPCR analysis of STAMP2 expression. The results are presented as boxplots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles, and the outlier is presented as an open circle. The statistical significance was determined by one-way ANOVA with a post hoc test. n = 3 in group week 0, 1, 2, and 4; n = 5 in refractory group. *P = 0.011; **P = 0.049. Error bars indicate SEM.
STAMP2 increases oxidative stress and is critical for prostate cancer. (2015)
Figure 6

STAMP2 affects ATF4 expression in PCa cells

A LNCaP cells were transfected with either control or STAMP2-specific siRNA in the presence of 10−8 M R1881. RNA was isolated, and qPCR was used to determine ATF4mRNA levels. Student's t-test was used to analyze the statistical significance, n = 3. *P = 0.002. Error bars indicate SD.

B Cell lysates from (A) were subjected to Western blot analysis.

C, D ATF4 expression in LNCaP (C) and 22Rv1 (D) cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by Western blot analysis.

E ATF4 expression in xenografted tumors of 22Rv1 cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by qPCR. Student's t-test was used to analyze the statistical significance, n = 4. *P = 0.022; **P = 0.015; ***P = 0.044. Error bars indicate SEM.

F CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. The association between ATF4 and STAMP2 in the tumor samples was determined by Western blot analysis.

G The MSKCC Prostate Oncogenome cDNA microarray dataset was obtained from the cBio Cancer Genomics Portal. Analysis was performed as described in Materials and Methods. The expression levels of STAMP2 and ATF4, as well as two ATF4 target genes (ASNS and SLC7A11), are presented.

Source data are available online for this figure.
STAMP2 increases oxidative stress and is critical for prostate cancer. (2015)
Figure 7

STAMP2 affects PCa cell growth through regulating ATF4 expression

A LNCaP cells were transfected with either control or ATF4 siRNA. Two days after transfection, cells were harvested and whole-cell lysates were made and used in Western blot analysis.

B LNCaP cells were transfected with either control or ATF4 siRNA and were cultured for 2 weeks. The colonies formed were stained and photographed.

C The area covered by the colonies on each plate in (B) was quantified and represented as percentage of the total area of the plate. *P = 0.0023.

D Cell lysates of LNCaP cells stably expressing an empty vector (Vector) or a vector expressing ATF4 (ATF4) were prepared and subjected to Western blot analysis with the indicated antisera.

E After transfection with the indicated siRNAs, cells from (D) were cultured for 2 weeks. The colonies formed were stained and photographed.

F The area of the colonies in (E) was quantitated using an image analysis system and represented as percentage of the total area of the plate. *P = 0.024.

G LNCaP cells were transfected with either control (Ctrl) or ASNS siRNA. The cells were then cultured for 2 weeks. The colonies formed were stained and photographed. Knockdown of ASNS was confirmed by Western blot analysis shown at the top.

H Quantification of the data shown in (G). *P = 0.0087.

Data information: In (C, F, and H), Student's t-test was used to analyze the statistical significance, n = 3. Error bars indicate SD. Source data are available online for this figure.
STAMP2 increases oxidative stress and is critical for prostate cancer. (2015)
Figure 8

ROS generation by STAMP2 depends on its ferrireductase activity

A 293T cells were transfected with plasmids expressing either an empty vector (Vec) or vectors expressing HA-tagged STEAP, STAMP1 (ST1), STAMP2 (ST2), or STAMP3 (ST3). Whole-cell extracts were prepared and subjected to Western blot analysis using anti-HA antibody or GAPDH as a loading control.

B Relative ferrireductase activities were measured in the cells expressing the different constructs used in (A) as described in Materials and Methods. *P < 0.0001.

C 293T cells were transfected with different plasmids expressing either an empty vector (Vec) or vectors expressing His-tagged wild-type STAMP2 (WT) or three STAMP2 mutants (dGSR, H304L, or H397L) as indicated. Whole-cell extracts were prepared and used in Western blot analysis with anti-His antibody or GAPDH antibody as a loading control.

D Relative ferrireductase activities were measured in the cells from (C) as described in Materials and Methods. *P = 0.0003.

E 293T cell line with Dox-inducible STAMP2 expression or vector control was generated. Western blot analysis confirmed STAMP2 expression in a Dox-inducible manner.

F The cells from (E) were either left untreated or treated with increasing amounts of Dox, and ferrireductase activity was determined. *P < 0.0001.

G The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Prior to ferrireductase activity measurement, the cells were treated with or without DPI as indicated for 1 h. *P < 0.0001; **P = 0.0012; ***P = 0.048.

H The cells from (E) were treated with 100 ng/ml Dox for 2 days. Oxidative stress was then measured by NBT staining, and the stained cells were photographed. There were equal numbers of cells on the plates for −/+ Dox as shown in Supplementary Fig S4.

I The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Then, the cells were treated with or without 10 μM DPI before being subjected to NBT staining. *P < 0.0001.

Data information: In (B, D, F, G, and I), Student's t-test was used to analyze the statistical significance, n = 3. Error bars indicate SD. Source data are available online for this figure.
STAMP2 increases oxidative stress and is critical for prostate cancer. (2015)
Figure 9

STAMP2 increases intracellular ROS by decreasing intracellular NADPH levels

A LNCaP cells stably expressing an empty vector (LN-Vec) or a vector expressing STAMP2 (LN-ST2) were established by lentivirus delivery. Cell lysates were prepared and subjected to Western blot analysis with the indicated antisera.

B Relative ferrireductase activities were measured in the cells described in (A). *P = 0.0003.

C NBT staining was performed in LNCaP cells stably expressing either an empty vector (LN-Vec) or a vector expressing wild-type STAMP2 (LN-ST2/WT) or a STAMP2 mutant (LN-ST2/dGSR) as indicated. The stained cells were then photographed and quantified. *P = 0.004.

D LNCaP cells were transfected with either scrambled siRNA (siCtrl) or siRNA against STAMP2 (siST2) and were cultured in the presence of 10 nM R1881 or vehicle for 2 days. Intracellular ROS levels were then measured by CellROX reagent staining. *P = 0.002. ns, not significant.

E LNCaP cells stably expressing an empty vector (Vec), a vector expressing wild-type STAMP2 (WT), or a STAMP2 mutant (dGSR) were cultured, and the NADPH/NADP+ ratio was determined as described in Materials and Methods. *P = 0.015.

F LNCaP cells were transfected with either control siRNA (siCtrl) or STAMP2 siRNA (siST2). Cells were then cultured for 2 days and harvested, and the NADPH/NADP+ ratio was determined as above. *P = 0.026.

G LNCaP cells stably expressing wild-type STAMP2 were treated with or without DPI (1 μM) for 4 h, harvested, and used in the NADPH/NADP+ assay. *P = 0.018.

Data information: Student's t-test was used to analyze the statistical significance, n = 3. Error bars indicate SD.
STAMP2 increases oxidative stress and is critical for prostate cancer. (2015)
Figure 10

Targeting STAMP2 in preclinical models of PCa results in tumor regression

A LNCaP cells were implanted subcutaneously into nu/nu mice. Once tumors reached 5 mm in size, mice (n = 5 per group) were given nanoliposome-encapsulated control siRNA or STAMP2 siRNA as described in Materials and Methods. Tumor volumes were measured at the indicated time points. *P = 0.001.

B Same experiment as in (A), but VCaP cells were used instead. *P = 0.001.

C A model for STAMP2 effects on growth and survival of PCa cells. Once activated, AR increases STAMP2 expression. STAMP2 catalyzes reduction of Fe3+ to Fe2+, also depleting NADPH levels, which increases ROS. Increased ROS then activate ATF4 expression which in turn activates target genes that adapt PCa cells to oxidative stress resulting in cell proliferation and survival.

Data information: Student's t-test was used to analyze the statistical significance between mice (n = 5 per group) treated with control siRNA vs STAMP2 siRNA. Error bars indicate SEM.