mouse monoclonal antibody raised against rpap3  (Cell Signaling Technology Inc)

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