Journal: bioRxiv

Article Title: The TRAPP complex mediates secretion arrest induced by stress granule assembly

doi: 10.1101/528380

Figure Lengend Snippet: (A) Cells were treated with 273 kinase inhibitors (10 μM) from the Sellekchem library and with SA, and Sec24C localization in G3BP puncta (% of total G3BP area) was calculated (see Materials and methods) and is reported as a percentage of the control (cells treated with SA alone). Gray dashed line, mean of control; gray box, control standard deviation (± 18.2%); red line, threshold of positive hits; red box: positive hits. (B) Enrichment Analysis of the positive hits; for each class of inhibitor, the enrichment (in percentage) of compounds falling into positive hits and the enrichment (in percentage) in the total number of compounds was calculated and expressed as a ratio. (C) Evaluation of CDK kinase activity upon SA treatment. Western blot analysis of phosphoSer-CDK substrates in control, SA (300μM, 30 min), Dinaciclib (10 μM, 180 min) and SA+Dinaciclib (150 min Dinaciclib and 30 minutes SA). CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition. Left, Western blot, right: Ponceau was used as loading control. Image, one representative experiments out of 3 independent replicates. (D) Drug specificity, from high (++++) to low (+). for the different CDKs as described by Selleckchem, and Sec24C recruitment to SGs as a percentage of control. (E) Analysis of Sec24C recruitment to SGs in control (MOCK), CDK1-KD, CDK2-KD and CDK1+2-KD HeLa cells. Data are expressed as percentage of Sec24C (mean intensity) in SG puncta normalized for cytosolic signal as a percentage of control conditions. Quantification of one representative experiment. N>100, mean ± s.e.m. ****p<0.0001. (F) Representative images of HeLa cells treated with the best three hits (Flavopiridol, SNS-032, Dinaciclib; 1μM 150 min) and then exposed to SA (300 μM, 30 min), followed by immunostaining for Sec24C and G3BP. Scale bar, 10μm. (G) HeLa cells were treated with Flavopiridol, SNS-032, or Dinaciclib for 150 min at the indicated concentrations, subsequently treated with SA (300 μM, 30 min), and then immunostained for Sec24C and G3BP and imaged by OPERA. BS-181, a specific CDK7 inhibitor, was used as negative control. Sec24C localization to SGs is expressed as a percentage of the control (cells treated with SA alone). Dashed red line, mean of control; yellow box, standard deviation of control (± 9.8%). (H) Cells were treated as in (G) and immunostained for TRAPPC2 and eIF3. Scale bar, 10 μm. The graph shows the quantification of TRAPPC2 localization in SG puncta (mean intensity). Data are mean ± s.e.m. expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol, SNS-032 or Dinaciclib treatment compared to the control (cells treated with SA alone). N=3, three independent experiments, n=60-80 cells per experiment. ****p<0.0001. . Evaluation of CDK1 and CDK2 knock down efficiency.

Article Snippet: To identify which specific CDK might be involved in Sec24C recruitment, we compared the potency of selected CDK inhibitors (Flavopiridol hydrochloride, SNS-032, Dinaciclib, AT7519, PHA-793887, ADZ5438, JNJ-7706621, PHA-767491, BMS-265246, PHA-848125, Roscovitine, Palbociclib, BS-181HCl) in the Sec24C recruitment assay with their ability to inhibit different CDK isoforms, as described by Selleckchem ( https://www.selleckchem.com/CDK.html ).

Techniques: Standard Deviation, Activity Assay, Western Blot, Activation Assay, Inhibition, Immunostaining, Negative Control

Journal: bioRxiv

Article Title: The TRAPP complex mediates secretion arrest induced by stress granule assembly

doi: 10.1101/528380

Figure Lengend Snippet: (A) Immunofluorescence images of Sec24C and TRAPPC2 in vehicle-treated and SNS-032-treated (1 μM, 3 h) cells. Scale bar, 10 μm. Graphs show the quantification of Sec24C and TRAPPC2 in the peri-Golgi area (mean intensity) normalized in SNS-032-treated cells relative to vehicle-treated cells (set as 100%). Mean ± s.e.m. of three independent experiments. **p<0.05, ****p<0.0001. (B) HeLa cells were treated with Dinaciclib (1 μM, 3 h) and permeabilized or not with digitonin as described in Materials and methods. A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency. Upper panels, non-permeabilized (NP) control cells; middle panels, permeabilized control cells; lower panels, permeabilized Dinaciclib-treated cells. Scale bar, 10 μm. (C) Quantification of Sec24C membrane association after CDK inhibitor treatment. Digitonin-permeabilized cells treated with vehicle (CTRL) or the indicated CDK inhibitor (1 μM, 3 h) were immunostained for Sec24C. The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug-treated cells is expressed as fold change compared to the control; n= 60-80; mean ± s.e.m. of three independent experiments ****p<0.0001. Scale bars, 10 μm.

Article Snippet: To identify which specific CDK might be involved in Sec24C recruitment, we compared the potency of selected CDK inhibitors (Flavopiridol hydrochloride, SNS-032, Dinaciclib, AT7519, PHA-793887, ADZ5438, JNJ-7706621, PHA-767491, BMS-265246, PHA-848125, Roscovitine, Palbociclib, BS-181HCl) in the Sec24C recruitment assay with their ability to inhibit different CDK isoforms, as described by Selleckchem ( https://www.selleckchem.com/CDK.html ).

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: The TRAPP complex mediates secretion arrest induced by stress granule assembly

doi: 10.1101/528380

Figure Lengend Snippet: (A) HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA (30 min, 300 μM), and immunostained for Sec24C or TRAPPC2 and eIF3. Scale bar 10 μm. (B) Analysis of the proliferation status of the cells after 8 h starvation in HBSS. Left, representative images of starved and nonstarved cells using EdU incorporation (see Materials and methods) and, right, quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells. (C) HeLa cells were seeded at different confluency, treated with SA and stained for Sec24C and G3BP as an SG marker. Scale bar, 10 μm. Flow cytometry (FACS) analysis (right panels) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency. The graph shows quantification of Sec24C mean intensity in SG puncta normalized for the cytosolic Sec24C at the indicated cell confluency. Mean ± s.e.m. of a representative experiment out of n=5 biological replicates. n= 50-80. ns, not significant; ****p<0.0001. (D) Growing and differentiated podocytes were treated with SA (300 μM, 30 min) and stained for TRAPPC2 or Sec24C and eIF3. Scale bar, 10μm. The graphs show quantification of TRAPPC2 and Sec24C (mean intensity) in SGs. Mean ± s.e.m. of three independents experiments ****p<0.0001. (E) CDK kinase activity in growing and differentiated podocytes. Western blot using a specific antibody recognizing phosphoSer-CDK substrates was used on total cell lysates. (F) Western blot analysis of phosphorylated retinoblastoma (p-RB) in growing (non-differentiated) versus differentiated podocytes. ß-actin was used as loading control. . Differentiation of podocytes.

Article Snippet: To identify which specific CDK might be involved in Sec24C recruitment, we compared the potency of selected CDK inhibitors (Flavopiridol hydrochloride, SNS-032, Dinaciclib, AT7519, PHA-793887, ADZ5438, JNJ-7706621, PHA-767491, BMS-265246, PHA-848125, Roscovitine, Palbociclib, BS-181HCl) in the Sec24C recruitment assay with their ability to inhibit different CDK isoforms, as described by Selleckchem ( https://www.selleckchem.com/CDK.html ).

Techniques: Staining, Marker, Flow Cytometry, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: The TRAPP complex mediates secretion arrest induced by stress granule assembly

doi: 10.1101/528380

Figure Lengend Snippet: (A) Box plot representing SG area (see Materials and methods) in mock, TRAPPC2- and TRAPPC3-depleted cells treated with SA. Distribution of values from three independent experiments. ****p<0.0001. (B) HeLa cells microinjected with control preimmune IgG or a TRAPPC3-specific antibody (right panels in green) were treated with SA. The TRAPPC3 Ab disrupts the Golgi , monitored using an anti-TGN46 Ab. Anti-G3BP was used to stain SGs. The graph shows quantification of the SG area. Mean ± s.e.m. three independent experiments; n>80. ****p<0.0001. Scale bar, 10 μm. (C) Structured Illumination Microscopy (SIM)-Super resolution (SR) images of endogenous TRAPPC2 and GFP-Sec23 localizing at SGs, stained for G3BP. Right, magnification of boxed area. Scale bar, 0.5 μm (D,E) Localization of Raptor (D) and RACK1 (E) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 μm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock. Mean ± S.D. three independent replicates. *p<0.02; **p<0.009 in (D) , *p<0.05; ****p<0.0001 in (E). (F,G) Localization of Raptor (F) and RACK1 (G) in untreated cells or cells pretreated with the indicated CDK inhibitor (1 μM, 150 min) and then with SA (300 μM, 30 min). G3BP was used to stain SGs. Scale bar, 10 μm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001. (H) Analysis of cell death after overnight recovery of HeLa cells treated or untreated with CDKi (SNS-032; Flavopiridol or Dinaciclib, 1 μM, 150 min) and then treated with SA (500 μM, 3 h). Images were automatically acquired by OPERETTA microscope. Values indicate the percentage of the total number of nuclei (stained with DAPI) positive for BoBo-3 staining. Mean ± s.d. of one representative experiment out of three independent replicates. ns, not significant, ****p<0.0001. . TRAPP depletion does not affect protein translation inhibition caused by SA treatment.

Article Snippet: To identify which specific CDK might be involved in Sec24C recruitment, we compared the potency of selected CDK inhibitors (Flavopiridol hydrochloride, SNS-032, Dinaciclib, AT7519, PHA-793887, ADZ5438, JNJ-7706621, PHA-767491, BMS-265246, PHA-848125, Roscovitine, Palbociclib, BS-181HCl) in the Sec24C recruitment assay with their ability to inhibit different CDK isoforms, as described by Selleckchem ( https://www.selleckchem.com/CDK.html ).

Techniques: Staining, Microscopy, Inhibition