Journal: Oral oncology

Article Title: DSG3 as a Biomarker for the Ultrasensitive Detection of Occult Lymph Node Metastasis in Oral Cancer Using Nanostructured Immunoarrays

doi: 10.1016/j.oraloncology.2012.08.001

Figure Lengend Snippet: A. Formalin fixed and paraffin embedded tissue sections of non-metastatic (N−) and metastatic lymph nodes (N+) show DGS3 expression only in N+, with the staining localized to the malignant squamous cells (n=30). All N− cases were negative (n=5). B. The epithelial specificity of DSG3 immunoreactivity was further confirmed using simultaneous cytokeratin (CK) staining. A representative example is shown, whereby the H&E stained tumor island is matched with CK and DSG3 expression, with no non-specific staining. An example of a N− case stained for DSG3 is shown.

Article Snippet: The following antibodies: goat-anti human DSG3 [AF1720]; mouse-anti human DSG3 [MAP1720], biotin labeled goat-anti human DSG3 [BAF1720], recombinant human DSG3 Fc Chimera protein [1720-DM], were from R&D Systems (MN, USA).

Techniques: Expressing, Staining

Journal: Oral oncology

Article Title: DSG3 as a Biomarker for the Ultrasensitive Detection of Occult Lymph Node Metastasis in Oral Cancer Using Nanostructured Immunoarrays

doi: 10.1016/j.oraloncology.2012.08.001

Figure Lengend Snippet: Normal oral mucosa biopsies and HNSCC were evaluated for DSG3 by IHC. A. DSG3 is expressed throughout the normal epithelium, but is stronger in the basal and parabasal layers. Diffuse expression was seen in the epithelial component of all HNSCC. B. Representative well (WD), moderate (MD) and poorly (PD) differentiated HSCC cases are shown. C. DSG3 was expressed in all tumors regardless of differentiation, with increased expression in WD cases. Numbers of cases analyzed is depicted. D. Total cell extracts from non-squamous (Jurkat, HMVEC, LEC, HUVEC) and oral-squamous (HN12, HN13, HN30, Cal27), and epidermal-squamous (HaCaT) were processed for Western blot analysis. Native DSG3 and its glycosylated forms were readily detected in squamous cells extracts, while absent from the non-squamous counterparts. These levels were compared with human recombinant DSG3 that was processed in a background of Jurkat cell lysate. Tubulin staining indicates equivalent loading and protein integrity.

Article Snippet: The following antibodies: goat-anti human DSG3 [AF1720]; mouse-anti human DSG3 [MAP1720], biotin labeled goat-anti human DSG3 [BAF1720], recombinant human DSG3 Fc Chimera protein [1720-DM], were from R&D Systems (MN, USA).

Techniques: Expressing, Western Blot, Recombinant, Staining

Journal: Oral oncology

Article Title: DSG3 as a Biomarker for the Ultrasensitive Detection of Occult Lymph Node Metastasis in Oral Cancer Using Nanostructured Immunoarrays

doi: 10.1016/j.oraloncology.2012.08.001

Figure Lengend Snippet: A. Multi-tumor TMAs (lung, breast, colon, prostate), and an oral specific TMA were assessed for DSG3 expression by IHC, and the staining scored for the presence of specific staining as (+) or (-). Most squamous cell lung cancers stained positive for DSG3, but very few of the adenocarcinomas gave positive reaction. All cores from the OSCC TMA scored positive. B. In lung cancer, DSG3 expression was positive in most squamous cell carcinomas (SCC) including lymph node metastasis (SCC Met), while few cases of adenocarcinomas (ADC) gave positive reaction, and all small cell lung carcinoma samples (SCLC), were negative.

Article Snippet: The following antibodies: goat-anti human DSG3 [AF1720]; mouse-anti human DSG3 [MAP1720], biotin labeled goat-anti human DSG3 [BAF1720], recombinant human DSG3 Fc Chimera protein [1720-DM], were from R&D Systems (MN, USA).

Techniques: Expressing, Staining

Journal: Oral oncology

Article Title: DSG3 as a Biomarker for the Ultrasensitive Detection of Occult Lymph Node Metastasis in Oral Cancer Using Nanostructured Immunoarrays

doi: 10.1016/j.oraloncology.2012.08.001

Figure Lengend Snippet: A. Scheme used for the ultrasensitive detection by the microfluidic immunoarray showing a single sensor in the array with capture DSG3 antibodies attached. Proteins are captured off-line on Ab2-magnetic bead (MB)-HRP bioconjugates, and after magnetic separation and washes, the MBs are injected into the immunoarray containing 8 sensors. A single immunoarray sensor is depicted. Following incubation, amperometric signals are generated by applying −0.3 V vs Ag/AgCl to the sensors by injecting a mixture of HRP-activator H2O2 and mediator hydroquinone (HQ). B. Varying recombinant DSG3 protein concentrations were used to generate a calibration plot. The sensitivity of DSG3 sensor using recombinant protein was 5646 nA mL [fg protein]−1 cm−2. C. Protein extracts of primary human oral squamous carcinomas (T 1–4) made with RIPA buffer were processed for detection of DSG3. High DSG3 levels were found to be present in all the samples, and this was confirmed by Western blot analysis of the same extracts for DSG3 (D). Tubulin was used as loading control.

Article Snippet: The following antibodies: goat-anti human DSG3 [AF1720]; mouse-anti human DSG3 [MAP1720], biotin labeled goat-anti human DSG3 [BAF1720], recombinant human DSG3 Fc Chimera protein [1720-DM], were from R&D Systems (MN, USA).

Techniques: Injection, Incubation, Generated, Recombinant, Western Blot, Control

Journal: Oral oncology

Article Title: DSG3 as a Biomarker for the Ultrasensitive Detection of Occult Lymph Node Metastasis in Oral Cancer Using Nanostructured Immunoarrays

doi: 10.1016/j.oraloncology.2012.08.001

Figure Lengend Snippet: H&E stained cryosections of representative non-metastatic (−) and metastatic (+) human cervical lymph nodes were scanned and the total number of tumor cells per section was quantified (Table 1). Serial sections of these lymph nodes were evaluated by immunofluorescence for DSG3 and detected only in metastatic lymph nodes (green). Vimentin (red) was used to identify stromal tissue, and nuclei of all cells were stained blue with DAPI (Fig. 5A). Protein extracts made from single cryosections of lymph nodes were used for the detection of DSG3 by Western blot analysis and DSG3 quantification using nanosensors. DSG3 levels were similar to background for all non-metastatic samples, while DSG3 levels in all metastatic cases were proportional to the number of invading HNSCC cells (Fig. 5B).

Article Snippet: The following antibodies: goat-anti human DSG3 [AF1720]; mouse-anti human DSG3 [MAP1720], biotin labeled goat-anti human DSG3 [BAF1720], recombinant human DSG3 Fc Chimera protein [1720-DM], were from R&D Systems (MN, USA).

Techniques: Staining, Immunofluorescence, Western Blot

Journal: Oral oncology

Article Title: DSG3 as a Biomarker for the Ultrasensitive Detection of Occult Lymph Node Metastasis in Oral Cancer Using Nanostructured Immunoarrays

doi: 10.1016/j.oraloncology.2012.08.001

Figure Lengend Snippet: DSG3 detection in metastastic lymph nodes Cryosections of non-metastatic (N−)(n=3) and metastatic (N+)(n=3) human cervical lymph nodes were collected and analyzed by nanosensor detection. The total number of tumor cells per cryosection was evaluated, and used to estimate the total amount of DSG3 per tumor cell.

Article Snippet: The following antibodies: goat-anti human DSG3 [AF1720]; mouse-anti human DSG3 [MAP1720], biotin labeled goat-anti human DSG3 [BAF1720], recombinant human DSG3 Fc Chimera protein [1720-DM], were from R&D Systems (MN, USA).

Techniques:

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: DSG2 is up-regulated in human cervical cancer and is associated with a poor prognosis. ( A ) Higher expression of DSG2 was found in cervical cancer samples than the normal tissues (based on TCGA database). ( B ) Kaplan–Meier plots of overall survival for cervical cancer samples with high/low DSG2 expression from the TCGA database.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: Knockdown of DSG2 in cervical cancer cells. ( A ) The knockdown efficiency of the three groups of siRNA in HeLa cells was 84%, 86% and 28%, respectively. ( B and C ) The specificity and validity of the siRNA knockdown of DSG2 expression in HeLa and SiHa cells was verified by qPCR ( B ) and WB ( C ). *P < 0.05, **P < 0.01.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Knockdown, Expressing

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: Knockdown of DSG2 inhibited proliferation and migration of cervical cancer cells. ( A ) The proliferation of HeLa and SiHa cells after knockdown of DSG2 was measured using CCK-8 assay. ( B ) The effect of DSG2 knockdown on cervical cancer cell clone formation. ( C ) The migration ability of HeLa and SiHa cells after knockdown of DSG2 was measured using Transwell assay. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Knockdown, Migration, CCK-8 Assay, Transwell Assay

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: ADAM17 is up-regulated in human cervical cancer and is associated with a poor prognosis. ( A ) The bioinformatics analysis of TCGA-CHOL dataset showed the positive expression correlation between DSG2 and ADAM17. ( B ) Higher expression of ADAM17 was found in cervical cancer samples than the normal tissues (based on TCGA database). ( C ) Kaplan–Meier plots of overall survival for cervical cancer samples with high/low ADAM17 expression from the TCGA database.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: DSG2 regulates the expression of ADAM17 in cervical cancer. ( A ) The effect of DSG2 knockdown on ADAM17mRNA expression was detected by qPCR. ( B ) The effect of DSG2 knockdown on ADAM17 protein expression was detected by WB. ( C ) The interaction between DSG2 and c-MYC was detected by Co-IP assay. ***P < 0.001.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Expressing, Knockdown, Co-Immunoprecipitation Assay

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: DSG2 regulates cervical cancer development by interacting with c-MYC. ( A ) HeLa cells were transfected with pcDNA(3.1)-DSG2 overexpression plasmid, and the mRNA level of DSG2 was detected by qPCR. ( B ) HeLa cells were transfected with pcDNA(3.1) overexpression plasmid, and the protein level of DSG2 was detected by WB. ( C and D ) DSG2 overexpressed HeLa cells were treated with a C-MYC inhibitor (10,058-F4, 50 μM), and the proliferative activity and migration ability were detected by CCK-8 ( C ) and clonal formation assay ( D ). ( E ) DSG2 overexpressed HeLa cells were treated with a C-MYC inhibitor, and ADAM17 protein expression was detected by WB.*P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Transfection, Over Expression, Plasmid Preparation, Activity Assay, Migration, CCK-8 Assay, Tube Formation Assay, Expressing

Journal: iScience

Article Title: OBP2A regulates epidermal barrier function and protects against cytotoxic small hydrophobic molecules

doi: 10.1016/j.isci.2024.111093

Figure Lengend Snippet:

Article Snippet: For immunostaining of OBP2A, filaggrin, involucrin, keratin 10, transglutaminase 1, claudin 1, corneodesmosin, and desmoglein 2, antibodies to OBP2A (orb37374, Biorbyt Ltd., Cambridge, UK), filaggrin (sc-66192, Santa Cruz Biotechnology, TX, USA), involucrin (ab53112, abcam, Cambridge, UK), keratin 10 (20R-CP002, Fitzgerald Industries International, MA, USA), transglutaminase 1 (sc-166467, Santa Cruz Biotechnology, TX, USA), claudin 1 (71–7800, Invitrogen, CA, USA), corneodesmosin (ab204235, abcam, Cambridge, UK), and desmoglein 2 (MAB947, R&D Systems, MO, USA) were used as primary antibodies, and donkey anti-rabbit IgG (Invitrogen, CA, USA) or donkey anti-guinea pig IgG (Jackson ImmunoResearch, PA, USA) was used as a second antibody.

Techniques: Imaging, Recombinant, Transfection, Saline, Sterility, Cell Culture, Protease Inhibitor, DC Protein Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Quantitation Assay, Fluorescence, Sequencing, Software

Journal: The Journal of Clinical Investigation

Article Title: Microenvironmental network of clonal CXCL13 + CD4 + T cells and Tregs in pemphigus chronic blisters

doi: 10.1172/JCI166357

Figure Lengend Snippet: ( A ) Skin biopsy sample showing tertiary lymphoid structures (TLSs) from a patient with PV ( , no. 3) stained with H&E and specific antibodies against CD20, CD4, CD138, and PNAd. Scale bar: 100 μm. ( B ) Schematic of the experiment. ( C ) Duration of skin blisters in TLS-negative ( n = 8) and TLS-positive ( n = 23) lesions. Student’s t tests were used to compare means for 2 groups. Data are shown as the mean ± SD. ** P < 0.005. ( D ) Representative immunofluorescence staining for DSG-specific B cells (triangles) and plasma cells (arrows). Tissues were stained with CD138 (red), CD20 (blue), rDSG1 or rDSG3 (green), and DAPI (light gray). Dotted lines indicate the margin of TLSs. Scale bar: 50 μm. PF, pemphigus foliaceus; PV, pemphigus vulgaris; rDSG1, recombinant desmoglein 1; rDSG3, recombinant desmoglein 3.

Article Snippet: Tissues were stained overnight at 4°C using the following primary antibodies: mouse anti-human CD20 (L26, Abcam); 6X His-recombinant human DSG1 and 6X His-recombinant human DSG3 (both from Cusabio); goat anti-human CXCL13 (R&D Systems); rabbit anti-human CCL5 (P230E, Thermo Fisher Scientific); rabbit anti-human CD138 (EP201), rabbit anti-human FDC (CNA.42), and mouse anti-human CD8 (C8/144B) (all from Cell Marque); rat anti-human CD4 (YNB 46.1.8, Santa Cruz); and mouse anti-human HLA-DR (L243, BioLegend).

Techniques: Staining, Immunofluorescence, Clinical Proteomics, Recombinant

Journal: bioRxiv

Article Title: Desmoplakin is a desmosomal mechanosensor

doi: 10.1101/2024.11.19.624364

Figure Lengend Snippet: (A) Schematic of desmosome structure. (B-D) Representative STED images of desmosomes showing a characteristic ‘railroad track’ pattern in (B) MCF7 WT cells, (C) MCF7 K19-KO cells, and (D) MCF7 K19-GfP cells. The desmosomes were immunolabeled for DPC (green) and Dsg2 (red). Scale bar: 500 nm. (E) The boxed area from the representative STED images of MCF7 WT cells shows a closer look at an individual DP railroad track with Dsg2 between the two parallel DP plaques. Scale bar: 200 nm. (F) Line-scan analysis of DPC and Dsg2 fluorescence intensity (indicated by the dashed line in E). (G) Quantification of DPC-DPC distance from WT and K19-KO cells showing that desmosomes in WT cells are wider than K19-KO cells. In all boxplots, the box represents the 25th and 75th percentiles with the median indicated and whiskers reach 1.5 times the interquartile range (IQR), defined as the difference between the 25th and 75th percentiles. Data points outside the whiskers are shown as outliers. Number of datapoints (n) = 630 (WT), 505 (K19-KO); Number of replicates (N) = 3. Mann-Whitney’s U test; ***, P<0.001. (H) Quantification of DPC-DPC distance in K19-KO, WT, and K19-GFP cells. Desmosomes in WT and K19-GFP cells are wider than K19-KO cells. n = 505 (K19-KO), 630 (WT), 716 (K19-GFP); N = 3. Kruskal-Wallis Test, followed by Dunn’s multiple comparison Test; ***, P<0.001.

Article Snippet: The following primary antibodies were used: human anti-DSG2 (MAB947, R&D systems), rabbit anti-DPC antibody (A303-356A, Bethyl Lab), rabbit anti-DPN antibody (25318-1-AP, Proteintech), chicken anti-GFP (600-901-215, Rockland), mouse anti-K19 antibody (A53-B/A2) (Santa Cruz Biotechnology), control mouse IgG (sc-2025) (Santa Cruz Biotechnology), and mouse anti-actin antibody (66009-1-Ig, Proteintech).

Techniques: Immunolabeling, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Desmoplakin is a desmosomal mechanosensor

doi: 10.1101/2024.11.19.624364

Figure Lengend Snippet: (A) Schematic of the desmosome under tension. The N-terminal of DP transitions from a closed conformation to an open conformation. (B) Quantification of desmosome half-unit widths (Dsg2-DPC distance) from WT and K19-KO cells. n = 1260 (WT), 1010 (K19-KO); N = 3. Mann-Whitney’s U test; ***, P<0.001. The distances between Dsg2 and DPC is significantly greater in the WT compared to the K19-KO cells. (C) Quantification of desmosome half-unit widths (Dsg2-DPN distance) from WT and K19-KO cells. n = 692 (WT), 826 (K19-KO); N = 3. Mann-Whitney’s U test; ns, P>0.05. The Dsg2-DPN distance in both cell lines are similar. (D) The mean values from the results in B and C are summarized in the bar chart to compare the DP length (DPN-DPC distance) between the WT and K19-KO cells. The DP length is 66 nm for the WT and 33 nm for the K19-KO, indicating that DP extends 33 nm in the WT cells. (E) Dispase assay after 24 h plating. The confluent cell sheets are treated with 4 mM EGTA for 1 h. The images show the intact cell sheets for WT, K19-KO, and K19-GFP rescued cells before stress and fragmented cell sheets after applying mechanical stress. (F) Quantification of the dispase assay from K19-KO, WT, and K19-GFP cells. n = 12 (KO), 12 (WT), 9 (K19-GFP); N = 3. Kruskal-Wallis Test, followed by Dunn’s multiple comparison Test; ***, P<0.001. The K19-KO cell sheets show a greater number of fragments compared to WT cell sheets, while K19-GFP cell sheets generate a similar number of fragments as WT. This indicates that elongation of DP strengthens intercellular adhesion in WT and K19-GFP cells.

Article Snippet: The following primary antibodies were used: human anti-DSG2 (MAB947, R&D systems), rabbit anti-DPC antibody (A303-356A, Bethyl Lab), rabbit anti-DPN antibody (25318-1-AP, Proteintech), chicken anti-GFP (600-901-215, Rockland), mouse anti-K19 antibody (A53-B/A2) (Santa Cruz Biotechnology), control mouse IgG (sc-2025) (Santa Cruz Biotechnology), and mouse anti-actin antibody (66009-1-Ig, Proteintech).

Techniques: Comparison

Journal: Anatolian Journal of Cardiology

Article Title: Desmosomal Junctions and Connexin-43 Remodeling in High-Pacing-Induced Heart Failure Dogs

doi: 10.14744/AnatolJCardiol.2023.2823

Figure Lengend Snippet: The Primers Used in this Study

Article Snippet: Then paraffin sections were incubated with the primary antibody [anti-Cx43 (ab11370, 1:400, Abcam, Cambridge, MA, USA), anti-DSG2 (bs-10152R, 1:200, Bioss, Beijing, China), and anti-DP (bs-1748R, 1:200, Bioss)] for 1 hour.

Techniques:

Journal: Anatolian Journal of Cardiology

Article Title: Desmosomal Junctions and Connexin-43 Remodeling in High-Pacing-Induced Heart Failure Dogs

doi: 10.14744/AnatolJCardiol.2023.2823

Figure Lengend Snippet: Immunofluorescent staining for the DP, DSG2, and Cx43. (A) Representative immunofluorescent images of DP, DSG2, and Cx43 in the myocardial. (B) Cx43 and DSG2 distribution. (C) Comparison of Pearson coefficient values for colocalization of Cx43 with various molecules (DSG2, DP) at the LM in the hearts of HF group. Cx43, Connexin-43; DP, Desmoplakin; DSG2, desmoglein-2; HF, heart failure; LM, lateral membrane.

Article Snippet: Then paraffin sections were incubated with the primary antibody [anti-Cx43 (ab11370, 1:400, Abcam, Cambridge, MA, USA), anti-DSG2 (bs-10152R, 1:200, Bioss, Beijing, China), and anti-DP (bs-1748R, 1:200, Bioss)] for 1 hour.

Techniques: Staining, Comparison, Membrane

Journal: Anatolian Journal of Cardiology

Article Title: Desmosomal Junctions and Connexin-43 Remodeling in High-Pacing-Induced Heart Failure Dogs

doi: 10.14744/AnatolJCardiol.2023.2823

Figure Lengend Snippet: Relative protein expression of DSG2, Cx43. Cx43, DSG2 protein expression by western blot. Cx43, Connexin-43; DSG2, desmoglein-2. ** P < .01 vs. control, *** P < .001 vs. control, n = 6 for each group.

Article Snippet: Then paraffin sections were incubated with the primary antibody [anti-Cx43 (ab11370, 1:400, Abcam, Cambridge, MA, USA), anti-DSG2 (bs-10152R, 1:200, Bioss, Beijing, China), and anti-DP (bs-1748R, 1:200, Bioss)] for 1 hour.

Techniques: Expressing, Western Blot, Control

Journal: Anatolian Journal of Cardiology

Article Title: Desmosomal Junctions and Connexin-43 Remodeling in High-Pacing-Induced Heart Failure Dogs

doi: 10.14744/AnatolJCardiol.2023.2823

Figure Lengend Snippet: Relative mRNA expression of DSG2, Cx43 by qRT-PCR. * P < . 05 vs. control, ** P < .01 vs. control, * ** P < .001 vs. control, n = 6 for each group.

Article Snippet: Then paraffin sections were incubated with the primary antibody [anti-Cx43 (ab11370, 1:400, Abcam, Cambridge, MA, USA), anti-DSG2 (bs-10152R, 1:200, Bioss, Beijing, China), and anti-DP (bs-1748R, 1:200, Bioss)] for 1 hour.

Techniques: Expressing, Quantitative RT-PCR, Control