EMBO/soda-nel huggingface dataset:
Anoctamin 8 Tethers Endoplasmic Reticulum and Plasma Membrane for Assembly of Ca2+ Signaling Complexes at the ER/PM compartment (2019)
Figure 3

Fig 3: ANO8 is required for maximal STIM1-Orai1 interaction and increases current inactivation under strong Ca2+ buffering.

(A, B) Knockdown of ANO8 (siA8) reduced CRAC current in cells transfected with Orai1 (O1) and STIM1 (S1) and buffered with 3 mM EGTA.

(C) Knockdown of ANO8 reduces the native store-dependent Ca2+ influx measured in store depleted cells by Ca2+ add-back.

(D,E) Knockdown of ANO8 reduced the number of store-dependent STIM1 puncta at the TIRF plane in cells expressing STIM1 and Orai1. Panel (D) shows representative images and (E) is the summary of 7 experiments.

(F, G) Current was measured with pipette solution contacting the fast and strong Ca2+ buffer 10 mM BAPTA in HEK cells transfected with STIM1, Orai1, and with (red) or without ANO8 (black). Panel (G) shows the increase in current density at peak current. Note the prominent current inactivation in the presence of ANO8.

(H) Knockdown of SARAF (red) in wild-type cells had no effect on current inactivation in the presence of 10 mM BAPTA.

(I) Knockdown of SARAF did not prevent the ANO8-dependent current inactivation in the presence of 10 mM BAPTA.

Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.
Anoctamin 8 Tethers Endoplasmic Reticulum and Plasma Membrane for Assembly of Ca2+ Signaling Complexes at the ER/PM compartment (2019)
Figure 4

Fig 4: Plasma membrane PI(4,5)P2 is required for ANO8 function.

(A, B) Depletion of plasma membrane PI(4,5)P2 with the FRB/FKBP system prevented the ANO8-mediated increase in STIM1-Orai1 current measured in pipette solutions containing 3 mM EGTA (A) or 10 mM BAPTA (B).

(C) HEK cells transfected with ANO8-YFP, STIM1-CFP Orai1-HA and FRB and mCherry-FKBP PI(4,5)P2 depleting constructs were treated with 0.2 µM rapamycin (rapa) for 5 min and then with 25 µM CPA for 10 min before fixation and imaging by confocal microscopy.

(D) Mutating ANO8 R948, R950, R951 in RPRRP, a region that is predicted to include an ANO8 PI(4,5)P2 binding site, prevented increased STIM1-Orai1 current and inactivation.

(E) Mutating ANO8 R949,950,951 to Q reduced translocation of ANO8.

Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.
Anoctamin 8 Tethers Endoplasmic Reticulum and Plasma Membrane for Assembly of Ca2+ Signaling Complexes at the ER/PM compartment (2019)
Figure 5

Fig 5: Inhibiting the SERCA pumps, activation of the IP3Rs and chelation of ER Ca2+ reduced ANO8-dependent current inactivation.

(A) Plot of the slope of inactivation as a function of current density in the presence (red) and absence of ANO8 (black). Increasing current density was obtained by varying external Ca2+ between 2-50 mM. The results are mean±s.e.m and difference were analyzed by unpaired t test.

(B, C) CRAC current was measured in HEK cells expressing STIM1 and Orai1 and in pipette solution containing 3 mM EGTA (b) or 10 mM BAPTA (c). SERCA pump was inhibited with 25 µM CPA in the red traces where indicated and 100 µM IP3 was included in the pipette solution (green in c).

(D, E) Experimental protocol and conditions as in (B and C) except that cells were also transfected with ANO8.

(F, G) HEK cells expressing STIM1, Orai1, and ANO8 were used to measure current in pipette solution containing 10 mM BAPTA and with (red) or without (black) 100 µM IP3 or 10 mM of the ER Ca2+ chelator TPEN (green). The current density (F) and the normalized current (G) are shown, illustrating the delay in current inactivation by IP3 and TPEN.

Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.
Anoctamin 8 Tethers Endoplasmic Reticulum and Plasma Membrane for Assembly of Ca2+ Signaling Complexes at the ER/PM compartment (2019)
Figure 6

Fig 6: ANO8 assembles Ca2+ signaling complexes at the ER/PM junctions.

(A, B): ER Ca2+ content was measured with ER-GECO1 in the periphery (A) and the center (B) or cells transfected with M3 receptors and with (red, green) or without (black, blue) ANO8. Receptor mediated store depletion was initiated by stimulating the cells with 0.5 mM carbachol in Ca2+-free solution. Cell stimulation was terminated with 10 µM atropine and ER Ca2+ uptake was initiated by perfusing the cells with a solution containing 5 mM Ca2+. The fluorescence was measured as F/F0 and normalized to the initial fluorescence in the presence of ANO8. Panel (C) shows traces of ER Ca2+ uptake at expanded time scale in the periods marked by rectangular in (A and B) and the averaged slopes of ER Ca2+ influx and (D) shows the averaged slope of Ca2+ release at the cell periphery (Peri) and cell center (Center). In (C) the traces were aligned along the Y axis to better show the difference in uptake rate.

(E) Enhancement by ANO8 of the Co-IP of expressed STIM1 with PMCA4a and SERCA2 and of Orai1 with SERCA2 in resting and store depleted cells.

(F) Effect of ANO8 expression on the Co-IP of the native SERCA2 and STIM1 in resting and store depleted cells. The columns are the mean±s.e.m of 3 experiments.

(G) FRET ratio between STIM1 and SERCA2 in response to store depletion and its enhancement by ANO8.

(H, I) Simultaneous measurements of ER Ca2+ with ER-GECO1 and PI(4,5)P2 hydrolysis with PHPLCδ-GFP in response to cell stimulation of M3 receptors. Traces are shown in (h) and the average slopes are in (i).

(J) Effect of ANO8 on the Co-IP of the native STIM1 and IP3Rs. The columns are the mean±s.e.m of 3 experiments.

(K) FRET ratio between expressed STIM1 and IP3R3 in response to store depletion and its enhancement by ANO8.

(L) FRET ratio between expressed STIM1 and PMCA4 in response to store depletion and its enhancement by ANO8.

Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.
Anoctamin 8 Tethers Endoplasmic Reticulum and Plasma Membrane for Assembly of Ca2+ Signaling Complexes at the ER/PM compartment (2019)
Figure 7

Fig 7: ANO8 tunes receptor-evoked Ca2+ signaling.

(A-D) HeLa cells transfected with GFP (black) or ANO8 (red) were stimulated with 0.5 and then 1 µM ATP to activate the native P2Y2 receptors. The oscillations were analyzed in terms of the % of responding cells (B), oscillation frequency (C), and the amplitude of the Ca2+ signal (D).

(E-H): HeLa cells were treated with scrambled siRNA (black) or siANO8 (red) and were stimulated with 1 and then 5 µM ATP. The oscillations were analyzed in terms of the % of responding cells (F), oscillations frequency (G), and the amplitude of the Ca2+ signal (H).

Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.
Lactate dehydrogenases promote glioblastoma growth and invasion via metabolic symbiosis (2022)
Figure 1

Figure 1: Regional expression of lactate dehydrogenases A and B in glioblastoma.

A. Left, 3D representation of an invading P3 spheroid embedded in a collagen matrix. Right, upper view: brightfield microscope image, lower view: coronal section of an invading spheroid. Scale bar: 100 µm.

B. Coronal section of a P3 spheroid embedded with paraffin and stained with DAPI (blue), LDHA (green), and LDHB (red). Magnification boxes show different areas as depicted in the main image. Quantification of LDHA and LDHB staining was performed on the spheroid areas as indicated in the graphs. Scale bar: 100 µm. Data are represented as mean ± s.d. (5-8 fields per area of interest, one representative spheroid of 3 independent invading spheroids)

C. Coronal section of P3 tumors in a mouse brain, embedded with paraffin and stained with DAPI (blue), LDHA (green), and LDHB (red). Magnification shows areas as depicted in the central illustration. Quantification of LDHA and LDHB staining was performed on the tumor areas as indicated in the graphs. Scale bar: 15 µm. Data are represented as mean ± s.d. (20 fields per area of interest, one representative brain of 2 independently implanted mouse brains) and analyzed using One-way ANOVA followed by Tukey's multiple comparisons test. LDHA: CC vs Core, P<0.0001; CC vs Periphery, P=0.18; Core vs Periphery, P<0.0001. LDHB: CC vs Core, P<0.0001; CC vs Periphery, P<0.0001; Core vs Periphery, P<0.0001.

D. Surface plots of the expression of LDHA, LDHB, CA9, and geneset enrichment for Hypoxia, in various stRNA samples (patients 1 to 3). The transparency of the spots is indicative of the expression/enrichment for the genes, with an additional layer of color based on expression/enrichment.

E. Spatially weighted correlation analysis of LDHA, LDHB, CA9, and geneset enrichment for Hypoxia, separated between the tumor core and the infiltrating areas.
Lactate dehydrogenases promote glioblastoma growth and invasion via metabolic symbiosis (2022)
Figure 5

Figure 5: LDHA/B knockout remodels mitochondrial functions and sensitizes tumors to radiation.

A. Western blot analysis of mitochondrial respiratory chain subunits from LDH KO P3 cells upon exposure to 21% or 0.1% O2. Roman numbers indicate the respiratory complex number. The diagram below represents densitometry quantification of the immunoblots normalized to vinculin and expressed as log2 foldchange to control cells in 21% and 0.1% O2 (n = 3 independent experiments).

B. Epifluorescence (top) and quantitative image analysis (bottom) of immune stained mitochondria (TOM20, green) from LDH KO P3 cells (Phalloidin, red; DAPI, blue) upon exposure to 0.1% O2 (n = 2 independent experiments, 28-32 cells per group). Data are represented as mean and analyzed using One-way ANOVA following by Tukey's multiple comparisons test. Mitochondrial mass: sgCont vs sgLDHA/B, P=0.005; sgLDHA/sgLDHB vs sgLDHA/B, p=0.006. Network aspect ratio: sgCont vs sgLDHA/B, P=0.03; sgLDHA vs sgLDHA/B, P=0.02; sgLDHB vs sgLDHA/B, p=0.04. Mitochondrial mass corresponds to the area covered by the mitochondria relative to the entire cell area. Network aspect ratio is the ratio of the major axis to minor axis of the mitochondrial network. Scale bar: 10 µm.

C. LDH KO P3 cell mass-specific respiration obtained by oxygraphy analysis (n = 4 independent experiments). Data are represented as mean ± s.e.m. and analyzed using Two-way ANOVA following by Tukey's multiple comparisons test: sgCont vs sgLDHA/B, P=0.015; sgLDHA/sgLDHB vs sgLDHA/B, P=0.005. For details, see Figure 2H.

D. Schematic representation of phenformin or irradiation effects on tumor cells.

E. Kaplan-Meier survival curves of xenotransplanted mice with LDHA/B KO (red) or control (blue) P3 cells, treated with phenformin or irradiated with 10 Gy (n = 6-8 mice per group). Data are analyzed using Log-rank (Mantel-Cox) test: sgCont Vehicle vs sgCont Phenformin, P=0.019; sgCont Vehicle vs sgCont Irradiation, P=0.0002; sgCont Irradiation vs sgLDHA/B Irradiation, P=0.024.
OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination (2016)
Figure 1

Figure 1. The de-ubiquitinase OTUB1 interacts with the RAS GTPases. A, A targeted MAPPIT screen identifies several DUBs as putative RAS interactors. A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait. pSEL(+2L)-HRAS G12V was expressed in HEK293T cells together with the indicated prey. BRAF served as a positive control. Each measurement was done in triplicate. The results are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells). The overall mean value + 2 s.d. served as a threshold. B, MAPPIT assay confirms the interaction between OTUB1 and RAS proteins. pSEL(+2L) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey. Empty vector and two random baits, MAL and eDHFR, were used as negative controls. REM2 and EFHA1 preys that bind to the bait receptor itself was used to evaluate expression of the RAS baits. The results are expressed as a mean of normalized luciferase activity ± s.e.m (leptin-treated cells vs leptin-untreated cells), n=3. C, NRAS Q61K mutant co-immunoprecipitates with OTUB1. At 48 hours post-transfection with Flag-tagged NRAS Q61K and HA-tagged OTUB1 expression constructs, HA-tagged OTUB1 was immunoprecipitated with anti-HA-agarose followed by immunoblotting using anti-Flag or anti-HA antibodies. D, OTUB1 interacts with wt NRAS and active NRAS mutant. Flag-tagged NRAS proteins were immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). E, GTP binding does not affect the complex formation between NRAS and OTUB1. Recombinant Flag-tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA-tagged OTUB1 in the excess of GTP-γ-S or GDP, followed by immunoblotting with the indicated antibodies. F, OTUB1 interacts with wt KRAS. Flag-tagged KRAS was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). C, D, E, F, Immunoprecipitates, IP. Whole cell lysate, WCL. G, OTUB1 co-localizes with KRAS at the plasma membrane. At 24 hours after co-transfection with GFP-tagged KRAS and HA–tagged OTUB1, HeLa cells were immunostained with anti-HA antibody. The outlined areas are shown at higher magnification at the top of each image. Scale bar, 10 micron.
OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination (2016)
Figure 2

Figure 2. OTUB1 triggers membrane localization of RAS ubiquitination by inhibiting its ubiquitination. A, Suppression of OTUB1 expression increases NRAS mono- and di-ubiquitination. 6xHis-tagged ubiquitin and Flag-NRAS were introduced into HEK293T cells expressing shGFP or shRNAs against OTUB1. Ubiquitinated NRAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody. B, C, D, Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination. 6xHis-tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA-OTUB1, the catalytically dead mutant HA-OTUB1 C91S, or empty vector (V). Ubiquitinated RAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody. E, OTUB1 induces membrane RAS re-localization. Confocal imaging of HeLa cells expressing the indicated constructs. For each sample, z-stacks obtained by scanning the sample from the apical to the basal layer. Step-size, 2 micron. F, RAS cellular distribution expressed as percentage of cells with specific RAS localization. For quantification of RAS localization, cells were randomly imaged using IN Cell Analyzer. RAS localization (>200 cells) was scored as intracellular and diffused (IN), mostly at the plasma membrane (PM), or both intracellular and plasma membrane (PM/IN). p-value=0.0005 as determined by Chi2-test, n=3. Representative images of HeLa cells expressing GFP-tagged KRAS are shown in Appendix Figure S1. Pull-down, PD. Whole cell lysate, WCL. Not modified, n.m.
OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination (2016)
Figure 3

Figure 3. OTUB1 increases RAS activity and enhances MAPK activation in wt RAS cells. A, B, OTUB1 overexpression promotes serum-induced activation of endogenous wt RAS (A) or wt NRAS (B). GTP-bound RAS was pulled down from HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) using recombinant RAF1 RBD conjugated to agarose beads. Input was controlled by immunoblotting using anti-panRAS or anti-Flag antibodies. C, D OTUB1 enhances serum-induced ERK1/2 phosphorylation in endogenous wt RAS (C) or wt KRAS (D). Levels of phosphorylated ERK1/2 and total ERK1/2 in HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m. n=2. E, F, Overexpression of wt OTUB1 (E) or the catalytically dead mutant OTUB1 C91S mutant (F) promotes serum-induced MAPK activation in cell expressing wt NRAS. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies. G, H, Overexpression of OTUB1 has no effect on serum-induced MAPK activation in cell expressing mutant NRAS Q61K or KRAS G12V. Whole cell lysates were analyzed by immunoblotting using antibodies as indicated. A,B,C,D,E,F,G,H Serum-starved HEK293T cells expressing the indicated constructs were stimulated with 10% serum for the indicated time periods. I, Immunoblot of OTUB1 overexpression in immortalized human embryonic kidney epithelial cells (HEK TEST), expressing empty vector (V), myristoylated AKT1 (myr-AKT), MEK1 D218,D222 mutant (MEKDD). J, K, OTUB1 cooperates with active AKT1 to promote anchorage-independent growth. Representative images of soft-agar colonies formed by HEK TEST cells expressing the indicated constructs. The number of soft agar colonies formed by cells expressing OTUB1 compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=2.
OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination (2016)
Figure 4

Figure 4. OTUB1 expression is up-regulated in wt KRAS lung tumors. A, Gains of OTUB1 and KRAS mutation are mutually exclusive in lung adenocarcinomas. OncoPrint showing the distribution of KRAS somatic mutations and OTUB1 copy number alterations in TCGA lung adenocarcinomas and squamous cell carcinomas obtained from cBioPortal (Cerami et al, 2012; Gao et al, 2013). Co-occurrence analysis showing significant mutual exclusivity between KRAS mutation and OTUB1 gain. B,C, OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13.1 copy number alteration. Pearson correlation of OTUB1 copy number (log2 ratio) with OTUB1 mRNA levels (RNAseq normalized read counts, log2 transformed) was analyzed. D,E,F, OTUB1 expression in TCGA lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (SCC) patients. Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Box whisker plots represent OTUB1 mRNA expression levels in TCGA lung carcinoma patients determined by RNAseq analysis. p-values were determined by two-sided t-Test. Total number of patients, n. G, Gain of 11q13.1 locus is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients with diploid or gain of the OTUB1 locus were stratified according tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test. H, OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients were stratified by tumor stages (T1-T4) and OTUB1 expression levels as described in Materials and Methods. Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test. I, KRAS mutation is a late event in lung adenocarcinoma progression. TCGA lung adenocarcinoma patients with different KRAS mutation status were stratified by tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.
OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination (2016)
Figure 5

Figure 5. OTUB1 is essential for tumorigenicity of lung cancer cells. A, Suppression of OTUB1 expression in NSCLC cell lines by specific shRNAs against OTUB1 was confirmed by immunoblotting using antibody against OTUB1. B, OTUB1 suppression decreases serum-induced ERK1/2 phosphorylation. Serum-starved A549 cells expressing shRNAs against OTUB1 or GFP were stimulated with 10% serum for the indicated time periods. Levels of phosphorylated ERK1/2 and total ERK1/2 were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m; p-values were determined by two-sided t-Test, n=2. C, D, OTUB1 suppression impairs the anchorage-independent growth of lung cancer cell lines. Representative images of soft-agar colonies formed by lung cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3. E, Xenograft tumor growth of A549 cells expressing shRNAs against GFP or OTUB1 subcutaneously injected into nude mice. p-value was determined by 2-way ANOVA.
OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination (2016)
Figure 6

Figure 6. OTUB1 promotes tumorigenicity of wt KRAS cells. A, OTUB1 overexpression promotes serum-induced MAPK activation in wt KRAS H1993 lung adenocarcinoma cells. B, OTUB1 overexpression does not affect serum-induced MAPK activation in mutant KRAS A549 lung adenocarcinoma cells. A,B, Serum-starved lung cells expressing Flag-OTUB1 or an empty vector (V) were stimulated with 10% serum for the indicated time periods. Whole cell lysates were analyzed by immunoblotting using antibodies against pERK1/2, ERK1/2, Flag and vinculin. C, Representative immunoblots of Flag-tagged OTUB1 overexpression in lung adenocarcinoma cell lines. D, E, OTUB1 overexpression affects anchorage-independent growth of lung adenocarcinoma cell lines. Representative images of soft-agar colonies formed by the indicated cells expressing Flag-OTUB1 or empty vector (V). The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3. F,G, Xenograft tumor growth of A549, H1993 or H1437 cells expressing Flag-OTUB1 or an empty vector (V) subcutaneously injected into nude mice. p-values were determined by 2-way ANOVA.