Journal: bioRxiv

Article Title: Structure of the RPAP3:TRBP interaction reveals an involvement of the HSP90/R2TP chaperone complex in dsRNA pathways

doi: 10.1101/2020.11.11.367672

Figure Lengend Snippet: Characterization of the TRBP:RPAP3 interaction. a . A candidate-based yeast two-hybrid (Y2H) screen performed in the yeast S. cerevisiae revealed an interaction between TRBP and RPAP3 in both orientations (left and middle panels). On the contrary, RPAP3 does not bind to PACT (right panel). The TRBP:Dicer interaction was used as a positive control. pAS2 and pACT2 plasmids expressed a protein fusion with the DNA binding domain or activation domain of transcription factor Gal4, respectively. The strength of the interactions was tested using increasing amounts of 3-amino-triazol (3-AT) from 0 to 40 mM. b . Co-expression experiments in E. coli . RPAP3 copurified with a hexahistidine His 6 -tagged version of TRBP on cobalt-based immobilized metal affinity chromatography (IMAC ) beads (TALON) at low salt concentration (LS), but not in high-salt conditions (HS). Single protein expression controls experiments for both untagged TRBP or RPAP3 proteins are shown in lanes 1-4. See materials and methods for details. c . Co-immunoprecipitation experiments performed in the T-Rex HEK293 cell line expressing a flagged version of TRBP upon doxycycline induction. ni: no doxycycline induction, i: doxycycline induction. RPAP3 is co-immunoprecipitated with the flagged TRBP protein. d . In cellulo Duolink assays performed in HeLa cells. Proximity ligation assay (PLA) reveals a close proximity of the endogenous TRBP and RPAP3 proteins in favor of their direct interaction. Nuclei were stained using DAPI, and cytoplasmatic actin using Alexa Fluor 546. Scale bar is 30 μm. See Materials and Methods for details.

Article Snippet: Antibodies and dilutions for IF and Duolink were the following: mouse monoclonal anti-RPAP3 at 1:250 dilution (Sigma-Aldrich, SAB407956); polyclonal rabbit anti-TRBP at 1:100 dilution (Abcam, ab72110); monoclonal mouse anti-Actin at 1:400 dilution (Abcam, ab3280); polyclonal rabbit anti-GAPDH at 1:750 dilution (Abcam, ab9485); monoclonal mouse anti-GAPDH at 1:750 dilution (Abcam, ab8245).

Techniques: Positive Control, Binding Assay, Activation Assay, Expressing, Affinity Chromatography, Concentration Assay, Immunoprecipitation, Proximity Ligation Assay, Staining

Journal: bioRxiv

Article Title: Structure of the RPAP3:TRBP interaction reveals an involvement of the HSP90/R2TP chaperone complex in dsRNA pathways

doi: 10.1101/2020.11.11.367672

Figure Lengend Snippet: Identification of the protein subdomains involved in the interaction. a . Representation of the subdomains of TRBP and RPAP3 as predicted with the software IUPRED and PSIPRED ( , ). Numbers correspond to the amino-acids residues located at each domain extremities. b . Co-expression experiments in E. coli performed with RPAP3, TRBP and their subdomains. For each protein pair, the first protein name on top of the gel always indicates the His 6 -tagged protein. Positive interactions are highlighted with red squares. C Two-hybrid screens performed in the yeast S. cerevisiae on TRBP, RPAP3 and their subdomains. The TRBP:Dicer interaction was used as a positive control. pAS2 and pACT2 plasmids respectively enable expression of a protein fusion with the DNA binding domain or activation domain of transcription factor Gal4. Strengths of the interactions were tested using increasing amounts of 3-amino-triazol (3-AT) from 0 to 40 mM. D . Co-expression experiments in E. coli performed with RPAP3 and TRBP subdomains. The TRBP and RPAP3 domains used are indicated below the gel lanes. The TRBP subdomains carried the His 6 -tag. “So”, “SnSo”, “Beads”, “MW” and “Pellet” design respectively the culture sonicate, supernatant sonicate, Talon beads, Molecular Weight marker and sonicate pellet.

Article Snippet: Antibodies and dilutions for IF and Duolink were the following: mouse monoclonal anti-RPAP3 at 1:250 dilution (Sigma-Aldrich, SAB407956); polyclonal rabbit anti-TRBP at 1:100 dilution (Abcam, ab72110); monoclonal mouse anti-Actin at 1:400 dilution (Abcam, ab3280); polyclonal rabbit anti-GAPDH at 1:750 dilution (Abcam, ab9485); monoclonal mouse anti-GAPDH at 1:750 dilution (Abcam, ab8245).

Techniques: Software, Expressing, Positive Control, Binding Assay, Activation Assay, Molecular Weight, Marker

Journal: bioRxiv

Article Title: Structure of the RPAP3:TRBP interaction reveals an involvement of the HSP90/R2TP chaperone complex in dsRNA pathways

doi: 10.1101/2020.11.11.367672

Figure Lengend Snippet: The same binding surface of TRBP seems to be involved for its interactions with Dicer or RPAP3. a . Co-expression experiments in E. coli of the three minimal protein subdomains involved in the TRBP:RPAP3 and TRBP:Dicer interactions. “So”, “SnSo”, “Beads”, “MW” and “Pellet” relate respectively to the culture sonicate, supernatant sonicate, Talon beads, Molecular Weight markers and sonicate pellet. b . Elution profile of the gel filtration assay performed on the 3 co-expressed TRBP, RPAP3 and TRBP minimal subdomains. c . Fractions collected were loaded onto a 10% SDS-PAGE.

Article Snippet: Antibodies and dilutions for IF and Duolink were the following: mouse monoclonal anti-RPAP3 at 1:250 dilution (Sigma-Aldrich, SAB407956); polyclonal rabbit anti-TRBP at 1:100 dilution (Abcam, ab72110); monoclonal mouse anti-Actin at 1:400 dilution (Abcam, ab3280); polyclonal rabbit anti-GAPDH at 1:750 dilution (Abcam, ab9485); monoclonal mouse anti-GAPDH at 1:750 dilution (Abcam, ab8245).

Techniques: Binding Assay, Expressing, Molecular Weight, Filtration, SDS Page

Journal: bioRxiv

Article Title: Structure of the RPAP3:TRBP interaction reveals an involvement of the HSP90/R2TP chaperone complex in dsRNA pathways

doi: 10.1101/2020.11.11.367672

Figure Lengend Snippet: Crystal structures of the TRBP:RPAP3 complex. a . Ribbon representation of the X-ray crystallographic structure of the TPR1 domain of RPAP3 and the dsRBD3 of TRBP at 1.49 Å resolution. RPAP3 (residues 133-249) and TRBP (residues 263-365) are drawn in green and orange, respectively. b . Ribbon representations of our crystal structure (left) and the one published by Wilson et al. (right) confirm that TRBP shares the same binding interface for its interaction with either Dicer or RPAP3.

Article Snippet: Antibodies and dilutions for IF and Duolink were the following: mouse monoclonal anti-RPAP3 at 1:250 dilution (Sigma-Aldrich, SAB407956); polyclonal rabbit anti-TRBP at 1:100 dilution (Abcam, ab72110); monoclonal mouse anti-Actin at 1:400 dilution (Abcam, ab3280); polyclonal rabbit anti-GAPDH at 1:750 dilution (Abcam, ab9485); monoclonal mouse anti-GAPDH at 1:750 dilution (Abcam, ab8245).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structure of the RPAP3:TRBP interaction reveals an involvement of the HSP90/R2TP chaperone complex in dsRNA pathways

doi: 10.1101/2020.11.11.367672

Figure Lengend Snippet: Two distinct surfaces of RPAP3 bind TRBP and the HSP90-tail peptide. a . Chemical shift perturbations of RPAP3-TPR1 upon binding of TRBP-dsRBD3. Backbone amide group resonances of the free and TRPB bound-states of RPAP3-TPR1 were compared using composite 1 H- 15 N chemical shifts (Δδ). Data were plotted againt the sequence of RPAP3. Position of helices was indicated in grey and the value corresponding to centile 80 was indicated with a dotted line. b . Residues in RPAP3 for which the Δδ value was superior to the centile 80 value were reported in green on the molecular surface of RPAP3-TPR1. The X-ray structure of the RPAP3-TPR1:TRBP-dsRBD3 complex was used. c . Superimposition of the crystal structure of RPAP3-TRBP (in green and orange) with the crystal structure of RPAP3 bound to the HSP90-tail peptide (in cyan) (PDB 4cgv). The HSP90-tail peptide (SRMEEVD) is shown as sticks. d . Protein co-expression assays in E. coli with the His 6 -TRBP-RPAP3 complex and human HSP70 or HSP90-MC. “So”, “SSo”, “Beads” and “Elu” relate respectively to the culture sonicate, supernatant sonicate, Talon beads and elution from the beads with imidazole. The co-purified proteins are indicated with colored arrows.

Article Snippet: Antibodies and dilutions for IF and Duolink were the following: mouse monoclonal anti-RPAP3 at 1:250 dilution (Sigma-Aldrich, SAB407956); polyclonal rabbit anti-TRBP at 1:100 dilution (Abcam, ab72110); monoclonal mouse anti-Actin at 1:400 dilution (Abcam, ab3280); polyclonal rabbit anti-GAPDH at 1:750 dilution (Abcam, ab9485); monoclonal mouse anti-GAPDH at 1:750 dilution (Abcam, ab8245).

Techniques: Binding Assay, Sequencing, Expressing, Purification

Journal: bioRxiv

Article Title: Structure of the RPAP3:TRBP interaction reveals an involvement of the HSP90/R2TP chaperone complex in dsRNA pathways

doi: 10.1101/2020.11.11.367672

Figure Lengend Snippet: Identification of key residues involved in TRBP:RPAP3 binding interface. a . Network of ionic interactions as well as hydrogen bonds between RPAP3 and TRBP. RPAP3 and TRBP are drawn in green and orange, respectively. b . Principle of the LUMIER-IP test. c . Bar plot showing LUMIER-IP co-efficiency for the interaction of Dicer with TRBP WT or Q357A. *** p-value <0,001 (Z-test comparing values of the FLAG IPs with eleven-times the mean value obtained in the control IP) or the interaction of RPAP3 D161A with TRBP WT or Q357A.

Article Snippet: Antibodies and dilutions for IF and Duolink were the following: mouse monoclonal anti-RPAP3 at 1:250 dilution (Sigma-Aldrich, SAB407956); polyclonal rabbit anti-TRBP at 1:100 dilution (Abcam, ab72110); monoclonal mouse anti-Actin at 1:400 dilution (Abcam, ab3280); polyclonal rabbit anti-GAPDH at 1:750 dilution (Abcam, ab9485); monoclonal mouse anti-GAPDH at 1:750 dilution (Abcam, ab8245).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structure of the RPAP3:TRBP interaction reveals an involvement of the HSP90/R2TP chaperone complex in dsRNA pathways

doi: 10.1101/2020.11.11.367672

Figure Lengend Snippet: TRBP and Dicer are functionally linked to the R2TP/HSP90 complex. a . Co-immunoprecipitation experiments performed using a transiently expressed V5-tagged TRBP protein, or the TRBP R354E. RPAP3, PIH1D1, RUVBL1 and RUVBL2 are the components of the R2TP complex in human. b . Bar plots representing the ratio RL/FFL to show the stability of RL-Dicer, RL-AGO1/2 and RL-TRBP WT/Q357A in 293T cell treated with DMSO or DMSO+ 2 µM of geldanamycin during 16 h. Plasmid expressing FFL alone is used to normalize the value obtained for RL protein in each assay. Phax and CSRP2 are two unrelated protein used as negative controls. * p-value <0,05. ** p-value <0,01 according to a Student test. % expression = 100*(RL-X GA /FFL GA ) / (RL-X DMSO /FFL DMSO ).

Article Snippet: Antibodies and dilutions for IF and Duolink were the following: mouse monoclonal anti-RPAP3 at 1:250 dilution (Sigma-Aldrich, SAB407956); polyclonal rabbit anti-TRBP at 1:100 dilution (Abcam, ab72110); monoclonal mouse anti-Actin at 1:400 dilution (Abcam, ab3280); polyclonal rabbit anti-GAPDH at 1:750 dilution (Abcam, ab9485); monoclonal mouse anti-GAPDH at 1:750 dilution (Abcam, ab8245).

Techniques: Immunoprecipitation, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Structure of the RPAP3:TRBP interaction reveals an involvement of the HSP90/R2TP chaperone complex in dsRNA pathways

doi: 10.1101/2020.11.11.367672

Figure Lengend Snippet: Hypothetical functions for the TRBP:RPAP3 interaction. See text for details.

Article Snippet: Antibodies and dilutions for IF and Duolink were the following: mouse monoclonal anti-RPAP3 at 1:250 dilution (Sigma-Aldrich, SAB407956); polyclonal rabbit anti-TRBP at 1:100 dilution (Abcam, ab72110); monoclonal mouse anti-Actin at 1:400 dilution (Abcam, ab3280); polyclonal rabbit anti-GAPDH at 1:750 dilution (Abcam, ab9485); monoclonal mouse anti-GAPDH at 1:750 dilution (Abcam, ab8245).

Techniques:

Journal: Nature Communications

Article Title: The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

doi: 10.1038/s41467-021-24792-4

Figure Lengend Snippet: a Schematic representation of R2TP with its four subunits (RPAP3, PIH1D1, and the RUVBL1/2 heterohexamer). RPAP3 is the core subunit that contacts directly HSP90, PIH1D1 and RUVBL1/2. b β galactosidase activity in Rpap3 wtsi/+ small intestines (top) and colon (bottom), as compared to negative controls ( n = 2). Scale bars = 50 μm are identical for all images. c IHC of Pih1d1 in the small intestine. Counter coloration of DNA with hematoxylin, with magnification (bottom). Top picture: bar represents 50 μm. Inset: arrows point to CBC stem cells, intercalated between Paneth cells with distinctive granules in the cytoplasm; bar is 20 μm. Micrograph is representative of n = 3. d Depletion of Rpap3 after tamoxifen injection. Western blots were revealed with antibodies against the indicated proteins in extracts of the jejunum (left) and the colonic (right) epitheliums of RPAP3 flox/flox controls (blue) or VilCreER T2 ; RPAP3 flox/flox animals (red), 5 days after the first tamoxifen injection. Each lane was loaded with the lysate obtained from a single animal and were verified for n = 12 small intestines and 8 colons, in three independent experiments. Molecular sizes are indicated on the right. e Individual weight variations in females (top panel) and males (bottom panel) of tamoxifen treated VilCreER T2 ; RPAP3 flox/flox animals (red curves, n = 6 females and n = 5 males) and RPAP3 flox/flox controls (blue curves, n = 8 females and n = 6 males). Individual weights were set at 100% for each animal at day 0, and analyzed by two-way ANOVA (genotype affects weights with p < 0.0001 in male and females) and Bonferroni’s post hoc multiple comparison tests (** p < 0.01; *** p < 0.001; **** p < 0.0001—see ). f Length of small intestines and colons from VilCreER T2 ; RPAP3 flox/flox mice (red points) and controls (blue points) measured at day 8 and day 10 of females (top, n = 3) and males (bottom, n = 4) were analyzed by two-tailed unpaired t test with Welch’s correction (females: * p = 0.0177, t = 4.750, df = 3; males: *** p = 0.0001, t = 14.14, df = 4). Statistics data represent individual assays with mean ± S.E.M. g Representative images of small intestine (white arrowheads) from VilCreER T2 ; RPAP3 flox/flox animals, which were filled with liquid (left panel) or blood (right panel) from day 8 to 10; a control organ is shown above. Source data are provided as a “ file”.

Article Snippet: TMA tissue sections were stained with a mouse monoclonal antibody raised against RPAP3 (proprietary 19B11 antibody at 1:10 dilution) or an antibody against human mTOR (clone 7C10, 1:100 dilution; overnight incubation; cat. number #2983; Cell Signaling).

Techniques: Activity Assay, Injection, Western Blot, Comparison, Two Tailed Test, Control

Journal: Nature Communications

Article Title: The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

doi: 10.1038/s41467-021-24792-4

Figure Lengend Snippet: a Schematic representation of the experimental setting: 8-week-old mice of the indicated genotype received two sequential injections of tamoxifen 24 h apart, and were analyzed 5 to 8 days after the first injection. 2 h before each sacrifice, BrdU was injected intraperitoneally to detect cells in S-phase (thin arrows). b Schematic representation of a crypt from the small intestine, with CBC stem cells (in green) sandwiched between Paneth cells (in brown) and progenitors forming the TA on top (purple). c Representative pictures of jejunum tissue sections stained with HE (top) or by IHC with anti-Ki67 antibody (bottom, Ki67 signal is brown) at indicated days after the first tamoxifen injection. Pink arrows point towards Paneth cells and black arrows towards CBC stem cells. Scale bar is shown in control HE panel. Each panel is representative of 8 to 12 animals analyzed in three independent experiments. Scale bar is identical for all panels and is 15 μm. d Representative pictures of jejunum taken from control (boxed in blue) and VilCreER T2 ; Rpap3 flox/flox animals (boxed in red), stained by IHC with anti-BrdU antibodies (brown arrows). n = 3–6 animals/ time point from two independent experiments. Scale bar is shown in control panel and is 50 μm. e Graph shows mean number of BrdU + cells/crypt in controls and VilCreER T2 ; Rpap3 flox/flox animals at day 7. Each point represents the average number of BrdU + cells calculated in n > 35 crypts from two different zones per animal ( n = 3). Mean values with S.E.M are indicated for each experimental group. Unpaired two-tailed t test with Welch’s corrections ( t = 11.66, df = 2) indicates significant difference between control and VilCreER T2 ; Rpap3 flox/flox animals ( p = 0.0073; n = 3). Source data are provided as a “ file”.

Article Snippet: TMA tissue sections were stained with a mouse monoclonal antibody raised against RPAP3 (proprietary 19B11 antibody at 1:10 dilution) or an antibody against human mTOR (clone 7C10, 1:100 dilution; overnight incubation; cat. number #2983; Cell Signaling).

Techniques: Injection, Staining, Control, Two Tailed Test

Journal: Nature Communications

Article Title: The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

doi: 10.1038/s41467-021-24792-4

Figure Lengend Snippet: a , b Staining for Olfm4 in the jejunum from control (top panel) and VilCreER T2 ; Rpap3 flox/flox animals 7 days ( a ) or 6 to 8 days after the first tamoxifen injection. Panels are representative for 2 to 4 animals/time point from at least two independent experiments. Scale bar is 20 μm in ( a ) and 50 μm in ( b ). c Schematic representation of a crypt from the small intestine, with CBC stem cells (in green) sandwiched between Paneth cells (in brown) and progenitors forming the TA on top (purple). d Representative micrographs of tissue sections immuno-stained for lysozyme, a specific marker of Paneth cells, in the jejunum of control (top) and VilCreER T2 ; Rpap3 flox/flox mice from day 6 to day 8. Panels are representative for 2 to 3 animals/time point from two independent experiments. Scale bar, identical in all pictures, is 50 μm. e Total number of apoptotic cells identified by cleaved caspase 3 (cleaved cas3 + ) per surface (mm 2 ) of jejunum for each mouse analyzed, at day 6. Mean values with S.E.M are indicated for each experimental group. Unpaired two-tailed t test with Welch’s correction indicates significant difference between controls and VilCreER T2 ; Rpap3 flox/flox animals ( p = 0.0384; t = 3.538, df = 3, n = 4). f Micrographs are tissue sections stained for cleaved caspase 3 in the jejunum. In control animals, cleaved caspase 3 + cells (brown arrows) are mainly detected at the tip of the villi, as a result of epithelial turnover (top panel). In the jejunum from VilCreER T2 ; Rpap3 flox/flox animals, at day 6 and 7, cleaved caspase 3 + cells were detected within the crypts (brown arrows). Panels are representative from 2 to 4 animals/time point, from two independent experiments. Scale bar, identical in all pictures, is 50 μm. Source data are provided as a “ file”.

Article Snippet: TMA tissue sections were stained with a mouse monoclonal antibody raised against RPAP3 (proprietary 19B11 antibody at 1:10 dilution) or an antibody against human mTOR (clone 7C10, 1:100 dilution; overnight incubation; cat. number #2983; Cell Signaling).

Techniques: Staining, Control, Injection, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

doi: 10.1038/s41467-021-24792-4

Figure Lengend Snippet: a Images are tissue sections of small intestines stained by immunohistochemistry (IHC) for Rpb1, the catalytic subunit of RNA polymerase II, from control Rpap3 flox/flox mice (blue frame, left panel), or VilCreER T2 ; Rpap3 flox/flox animals at day 6 (red frame, right panel), with magnifications of crypts (scale bars used for the two magnification insets are identical between blue and red frames). Note that the staining in stromal cells is nuclear in both wild-type and VilCreER T2 ; Rpap3 flox/flox animals (stromal cells do not express the Cre) and control epithelium, while it becomes cytoplasmic in the mutant epithelium. Panels are representative for n = 6 animals from three independent experiments. Scale bar is 50 and 10 μm for insets, as shown in control panels. b Schematical interpretation of the micrographs in ( a ). In control epithelial cells (blue), R2TP incorporates Rpb1 into RNA PolII, which is then imported into the nucleus. In the absence of Rpap3 (red), neo-synthesized Rpb1 accumulates in the cytoplasm. c , d Western blot analysis of preparations enriched for epithelial crypt cells from the jejunum of animals, sacrificed 6 days after the first tamoxifen injection. NOP58, EFTUD2, and PRPF8 ( c ), mTOR, ATM, ATR, and TRRAP ( d ) were detected with specific antibodies. Tubulin and GAPDH were used as loading controls. Quantification of the signal ratios are indicated on top of each lane (average for the control ratios was arbitrarily set to 100). Each lane was loaded with the lysate obtained from one animal of the indicated genotype ( n = 3 per genotype). Similar results were obtained with animals from at least two independent experiments. Apparent molecular weights are indicated on the right. Source data are provided as a “ file”.

Article Snippet: TMA tissue sections were stained with a mouse monoclonal antibody raised against RPAP3 (proprietary 19B11 antibody at 1:10 dilution) or an antibody against human mTOR (clone 7C10, 1:100 dilution; overnight incubation; cat. number #2983; Cell Signaling).

Techniques: Staining, Immunohistochemistry, Control, Mutagenesis, Synthesized, Western Blot, Injection

Journal: Nature Communications

Article Title: The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

doi: 10.1038/s41467-021-24792-4

Figure Lengend Snippet: a Micrographs are tissue sections stained for p53 by immunofluorescence in controls (top) and VilCreER T2 ; Rpap3 flox/flox mice (bottom) at day 6, representative of n = 7 animals from three independent experiments. Nuclei were stained with DAPI. Scale bar is 50 μm and is identical for all pictures. b Micrographs are tissue sections stained by immunofluorescence for p53 (Cy5, red) and lysozyme (Alexa 488, green), a marker of Paneth cells, in VilCreER T2 ; Rpap3 flox/flox mice at day 6 ( n = 4). Nuclei were stained with DAPI. Scale bar, 50 μm, is identical for all pictures. c Pictures of jejunum sections stained with HE (top) or by IHC with anti-Ki67 antibodies (bottom) in P53 KO ( VilCreER T2 ; Rpap3 flox/+ ; Trp53 flox/flox ), Rpap3 KO (VilCreER T2 ; Rpap3 flox/flox ; Trp53 flox/+ ) and double Rpap3 KO; P53 KO (VilCreER T2 ; Rpap3 flox/flox ; Trp53 flox/flox ) mice at day 6 to 8 after the first tamoxifen injection. Pictures are representative for each single KO ( n = 3) and double KO ( n = 5–7) mice, from three independent experiments. Scale bar, 50 μm, is identical for all pictures. d Pictures of jejunum sections stained by IHC with anti-Rpb1 antibody in control, P53 KO ( VilCreER T2 ; Rpap3 flox/+ ; Trp53 flox/flox ), Rpap3 KO (VilCreER T2 ; Rpap3 flox/flox ; Trp53 flox/+ ) and double Rpap3 KO; P53 KO (VilCreER T2 ; Rpap3 flox/flox ; Trp53 flox/flox ) mice at day 6 following tamoxifen injection. Pictures are representative of n = 3 for each single KO, n = 7 for double KO, from two different experiments. Scale bar, 50 μm, is identical for all pictures. e Total number of apoptotic cells at day 6 per surface (mm 2 ) of the jejunum for each mouse analyzed. n = 2 for wild type, n = 3 for each single KO, n = 7 for double KO, from two different experiments. Mean values with S.E.M are indicated for experimental groups with n > 3. One-way ANOVA analysis ( p = 0.0006) with Bonferroni’s multiple comparison post-test (** p < 0.001). Source data are provided as a “ file”.

Article Snippet: TMA tissue sections were stained with a mouse monoclonal antibody raised against RPAP3 (proprietary 19B11 antibody at 1:10 dilution) or an antibody against human mTOR (clone 7C10, 1:100 dilution; overnight incubation; cat. number #2983; Cell Signaling).

Techniques: Staining, Immunofluorescence, Marker, Injection, Control, Comparison

Journal: Nature Communications

Article Title: The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

doi: 10.1038/s41467-021-24792-4

Figure Lengend Snippet: a Representative micrographs of colon sections stained by Periodic Acid Schiff stain (PAS, for labeling of goblet cells), IHC for Ki67 and CD44v6 (a marker of the colonic stem cells) and IF for p53, in controls Rpap3 flox/flox (left) and VilCreER T2 ; Rpap3 flox/flox mice (right) at day 8 ( n = 4 from at least two independent experiments). Scale bars are 50 μm. b Western blot analysis of colonic epithelial cells from animals, sacrificed 7 days after the first tamoxifen injection. mTOR, ATM, and tubulin were detected with specific antibodies. Quantification of the signal ratios are indicated on top of each lane (average for the control ratios was arbitrarily set to 100). Each lane was loaded with the lysate obtained from one animal of the indicated genotype (representative for n = 5 KO animals in one experiment). Molecular weights are indicated on the right. c Micrographs are tissue sections stained by IHC for cleaved caspase 3 in Rpap3 flox/flox (blue) and VilCreER T2 ; Rpap3 flox/flox (red) mice at day 6, representative for n = 4 from two different experiments. Total number of apoptotic cells at day 8 per surface (mm 2 ) of the jejunum for each mouse analyzed. Mean values with S.E.M are indicated for each experimental group. Unpaired two-tailed t test with Welch’s correction indicates significant difference between controls and VilCreER T2 ; Rpap3 flox/flox animals ( p = 0.0234, t = 2.625, df = 5). Scale bar is 50 μm. d Picture of organoids cultures. Crypts from small intestine (top) and colon (bottom) were prepared from Rpap3 flox/flox (blue) and VilCreER T2 ; Rpap3 flox/flox (red) mice 3 days after tamoxifen injection ( n = 3). Identical number of crypts were seeded and organoids culture were monitored every day. Organoids from control animals started budding after 72 h in culture for small intestine crypts and 96 h for colonic crypts. Organoids generated from VilCreER T2 ; Rpap3 flox/flox animals degenerated in culture before budding. Scale bar are 100 μm for all pictures and 20 μm for organoid insets. Source data are provided as a “ file”.

Article Snippet: TMA tissue sections were stained with a mouse monoclonal antibody raised against RPAP3 (proprietary 19B11 antibody at 1:10 dilution) or an antibody against human mTOR (clone 7C10, 1:100 dilution; overnight incubation; cat. number #2983; Cell Signaling).

Techniques: Staining, Labeling, Marker, Western Blot, Injection, Control, Two Tailed Test, Generated

Journal: Nature Communications

Article Title: The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

doi: 10.1038/s41467-021-24792-4

Figure Lengend Snippet: a Schematic representation of the experimental setting. Eight-week-old Lgr5-GFP-IRES-CreER T2 ; Rpap3 flox/flox mice received five sequential intra-peritoneal injections of tamoxifen and were analyzed 7 or 10 days after the first injection. In this genetic model, the Cre is expressed in the Lgr5 + CBC stem cells labeled by GFP (see scheme on the right). b Representative images of tissue sections labeled by immunofluorescence with antibodies against GFP (green) and Rpb1 (red), with DAPI counter-staining of nuclei (blue). Please note the mosaic expression of GFP. Panels are representative for 5 animals/time point from two independent experiments. White arrow: GFP + crypts. Asterisks: GFP − crypts. Scale bars (40 or 20 μm) are identical for matching panels. c Images are intestine tissue sections of wild-type animals stained for BrdU. Animals received one BrdU injection and were sacrificed at the indicated time point. The experiment was repeated twice (2 to 4 animals/time point from two different experiments). Scale bars (50 μm) are identical for all pictures.

Article Snippet: TMA tissue sections were stained with a mouse monoclonal antibody raised against RPAP3 (proprietary 19B11 antibody at 1:10 dilution) or an antibody against human mTOR (clone 7C10, 1:100 dilution; overnight incubation; cat. number #2983; Cell Signaling).

Techniques: Injection, Labeling, Immunofluorescence, Staining, Expressing

Journal: Nature Communications

Article Title: The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

doi: 10.1038/s41467-021-24792-4

Figure Lengend Snippet: a The graph depicts transcript levels of R2TP components in human primary colorectal tumor samples ( n = 380), as compared to normal solid tissues ( n = 51) from COADREAD cohort. y -axis: Log2 normalized counts for the indicated transcript. Distributions are presented as box-and-whisker plots (center line: median; box limits, first and third quartiles; whiskers, 10th and 90th percentiles). Statistical significance was determined by one-way ANOVA (* p < 0.001). b Kaplan–Meier analysis of disease-free survival among 177 CRC patients according to the proportion of RPAP3-expressing cells in tumor tissues. Solid green line and dashed blue line indicate high and low proportion of RPAP3-expressing tumoral cells, respectively. Statistical significance was determined by log-rank test ( P = 0.037). Right panels show examples of CRC tissues with low (top) or high (bottom) RPAP3 expression, with scale bar. c Proposed model for R2TP activity in the small and large intestine. R2TP assembles cellular machineries such as RNA polymerases, snoRNPs, snRNPs, and PIKKs-complexes in CBCs and progenitors in the proliferative compartment (blue cells). Differentiated cells (including Paneth cells from the small intestine crypts, in pink) mostly rely on the complexes assembled during the proliferative phase. A defect in R2TP activity induces client dysfunction, cell cycle arrest and apoptosis via p53, and eventually, epithelium degradation.

Article Snippet: TMA tissue sections were stained with a mouse monoclonal antibody raised against RPAP3 (proprietary 19B11 antibody at 1:10 dilution) or an antibody against human mTOR (clone 7C10, 1:100 dilution; overnight incubation; cat. number #2983; Cell Signaling).

Techniques: Whisker Assay, Expressing, Activity Assay

Journal: bioRxiv

Article Title: The R2TP chaperone assembles cellular machineries in intestinal CBC stem cells and progenitors

doi: 10.1101/2019.12.19.882712

Figure Lengend Snippet: (A) Western blot analysis of preparations enriched for epithelial crypt cells from the jejunum of animals, sacrificed 6 days after the first tamoxifen injection. NOP58, PRP8, ATR and mTOR and TRRAP were detected with specific antibodies. Tubulin and GAPDH were used as loading controls. Each lane was loaded with the lysate obtained from one animal of the indicated genotype (6 animals per gel). Molecular weights are indicated on the right. See Supplemental Figure 4 for an expanded view of the membranes and signal quantification. (B) Images are tissue sections of small intestines stained by immunohistochemistry (IHC) for Rpb1, the large subunit of RNA polymerase II, from control Rpap3 flox/flox mice (blue frame, left panel), or VilCreER T2; Rpap3 flox/flox animals at day 6 (red frame, right panel). Note that the staining in stromal cells is nuclear in both wild-type and VilCreER T2 ; Rpap3 flox/flox animals (stromal cells do not express the Cre), while it becomes cytoplasmic in the mutant epithelium. The scale is identical for both pictures. Panels are representative for n=6 animals from three independent experiments. (C) Magnification of crypts from (B). The scale bar is identical for both images. (D) Schematic interpretation of the IHC in (B, C). In control epithelial cells, R2TP incorporates Rpb1 into RNA PolII, which is then imported into the nucleus. In the absence of Rpap3, neo-synthesized Rpb1 accumulates in the cytoplasm.

Article Snippet: Primary antibodies from Cell Signaling: ATM D2E2 (CST 2873S) rabbit at 1/1000, ATR E1S3S (CST 13934S) rabbit at 1/1000, mTOR (CST 2972S) rabbit 1/1000, p53 1C12 (CST2524S) mouse 1/1000; PRP8 (sc-30207, Santa Cruz) rabbit 1/200; from ABCAM: EFTUD2 (ab72456) rabbit 1/2000, GAPDH (ab8245 6C5) mouse 1/10000; and from Sigma: NOP58 (HPA018472) rabbit 1/100, RPAP3 (SAB1411438) rabbit 1/1000, Tubulin (I2G10) mouse 1/500, TRRAP 2TRR-1B3 (MABE1008) mouse 1/1000.

Techniques: Western Blot, Injection, Staining, Immunohistochemistry, Control, Mutagenesis, Synthesized