hsp70 inhibitor ver 155008  (TargetMol)

Journal: Cell Communication and Signaling : CCS

Article Title: Hsp70s regulate circadian rhythm by interacting with PERIOD

doi: 10.1186/s12964-026-02805-3

Figure Lengend Snippet: Hsp70s Regulate Circadian Rhythm under various conditions. A Immunoprecipitation assays were performed to compare the interaction strength between wild-type PER and Hsp70Ba under various temperature conditions. Western blot analysis was performed with mouse anti-V5 (ABclonal, Cat#AE017), mouse anti-HA (ABclonal, Cat#AE008) antibodies and rabbit anti-Actin (ABclonal, #AC026). B Statistics of ( A ). N = 3. Statistical differences were assessed using one-way ANOVAs and post-hoc Tukey tests (P < 0.05). C Representative double plot actograms showing average locomotor activity for the respective genotypes under heat shock conditions. White background indicates light, and black indicates darkness. D Analysis of circadian rhythm persistence in Hsp70 mutants and w 1118 controls following heat shock. P-values from two-sided Fisher’s exact test are indicated (***P < 0.001)

Article Snippet: For drug treatment, 100 μM of the Hsp70 inhibitor VER-155,008 (Target Mol, #T7010) was dissolved in the media.

Techniques: Immunoprecipitation, Western Blot, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Hsp70s regulate circadian rhythm by interacting with PERIOD

doi: 10.1186/s12964-026-02805-3

Figure Lengend Snippet: Hsp70 Protein Interacts with PER, Affecting PER Phosphorylation and Cellular Localization. A Relative per expression at different time points in Hsp70 mutants and the control group. B Antibody specificity evaluation in western blot analysis for PER protein. C PER protein detection by western blots at ZT3, ZT9, ZT15, and ZT21. B - C Western blot analysis was performed with anti-PER (a gift from Dr. Jeffrey Price’s laboratory at the University of Missouri-Kansas City, U. S.) and mouse anti-β-Tubulin (ABclonal, Cat#AC021). D Quantification of results in ( C ). N = 5. Statistical differences were measured using an unpaired Student’s t-test. **P < 0.01, ***P < 0.001. E Statistics of the cellular localization of PER in s-LNvs and l-LNvs clusters of clock neurons in indicated genotypes. C represents cytoplasm, and N represents nucleus. F Immunoprecipitation experiments to detect the interaction between PER, TIM, and Hsp70s. Western blot analysis was performed with mouse anti-V5 (ABclonal, Cat#AE017), mouse anti-HA (ABclonal, Cat#AE008) antibodies and mouse anti-β-Tubulin (ABclonal, Cat#AC021). Samples were prepared in S2 cells, with β-tubulin used as an internal control. G Calculation of average TIM binding by PER molecule. N = 3. Significance of differences was determined using one-way ANOVAs and post-hoc Tukey tests. H Immunoprecipitation experiments to detect the interaction between PER and Hsp70s in tim 01 and w 1118 controls. Western blot analysis was performed with anti-PER (a gift from Dr. Jeffrey Price’s laboratory at the University of Missouri-Kansas City, U. S.) and anti-β-Tubulin (ABclonal, Cat#AC021). β-tubulin was used as an internal control. ( I ) Calculation of average Hsp70 binding by PER molecule. N = 5. The t-test was used to calculate the significance of differences. **P < 0.01

Article Snippet: For drug treatment, 100 μM of the Hsp70 inhibitor VER-155,008 (Target Mol, #T7010) was dissolved in the media.

Techniques: Phospho-proteomics, Expressing, Control, Western Blot, Immunoprecipitation, Binding Assay

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: Intermittent heat stimulation training improves high temperature tolerance in mice. A. Flowchart of the experiment to improve heat tolerance in mice through intermittent heat stimulation training. B & C. ELISA was used to analyze the plasma concentrations of cytokines (IL-1β and TNF-ɑ) in control, HS, and HS+HA group mice (n =8) 16 h after HS. D. WB was used to detect the expression of HIF-1α and HSP70 protein. β-actin was used as an internal control. E. IF analysis of isolated microvessels stained with antibodies for each cell component. Endothelial markers (CD31, green channel; VWF, red channel) were detected in all microvessel fragments. Scale bar = 50 μm. F. IF was used to detect apoptosis in isolated microvessels stained with TUNEL. Scale bar = 100 μm. **** P < 0.0001.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, Expressing, Isolation, Staining, TUNEL Assay

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: HA induces tolerance to high temperatures via the HIF-1α/HSP70 signaling. A & B. The bEnd.3 cells were exposed to 40℃ for 2 h daily, continuously for 1 day, 3 days, and 5 days. RT-qPCR was used to detect the expression of Hif-1α and Hspa1a mRNA. C. WB was used to detect the expression of HIF-1α, HSP90AA1, and HSP70 protein. β-actin was used as an internal control. D. IF was used to detect the expression of HIF-1α and HSP70 protein. E. WB was used to detect the expression of HIF-1α and HSP70 protein. β-actin was used as an internal control. F. bEnd.3 HA cells were exposed to HS for 4 h and treated with LW6, VER-155088, or a combination of both. Cell viability was detected using the CCK-8 assay. G. bEnd.3 HA cells were exposed to HS for 4 h and treated with si- Hif-1α , si- Hspa1a , or a combination of both. Cell viability was detected using the CCK-8 assay. H. Flow cytometry with Annexin V and PI staining was used to determine the apoptotic index of cells. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Quantitative RT-PCR, Expressing, Control, CCK-8 Assay, Flow Cytometry, Staining

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: HA enhances HIF-1α protein stability via HSP70 in bEnd.3 cells. A. bEnd.3 and bEnd.3 HA cells were treated with CHX for the indicated times and analyzed by WB. B. IF analysis of HIF-1α subcellular localization. Nuclei are stained with DAPI (blue). n = 5 per group, scar bar = 50 μm. C. bEnd.3 HA cells were treated with DMSO, si-NC, VER-155008, or si- Hspa1a and with CHX for 6 h. WB was used to detect the HIF-1α protein expression. D. IF was used to detect the HIF-1α protein expression. Nuclei are stained with DAPI (blue). n=5 per group, scar bar = 50 μm. E. bEnd.3 and bEnd.3 HA cells were treated with si-NC, VER-155008, or si- Hspa1a . The extracts were immunoprecipitated with anti-HIF-1α antibodies and immunoblotted with anti-ubiquitin, anti-HIF-1α, and anti-HSP70 antibodies. β-actin was used as an internal control.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Staining, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Control

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: HA promotes the interaction between HIF-1α and HSP70 proteins in bEnd.3 cells. A. ZDOCK prediction was used to model the predicted interaction between HSP70 and HIF-1α. B. Predicted ZDOCK binding score, hydrogen bonds, and electrostatic interactions with amino acid sites between HSP70 and HIF-1α. C. Colocalization of HIF-1α (red) with HSP70 (green) in bEnd.3 and bEnd.3HA cells. Nuclei are stained with DAPI (blue). n=5 per group, scar bar = 50 μm. D & E. bEnd.3 and bEnd.3HA cells were treated with si-NC, si-Hif-1α, or si-Hspa1a. The extracts were immunoprecipitated with anti-HIF-1α or anti-HSP70 antibodies and immunoblotted with anti-HIF-1α and anti-HSP70 antibodies.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Binding Assay, Staining, Immunoprecipitation

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: HA induces tolerance to hypoxia via the HIF-1α/HSP70 signaling. A. bEnd.3 and bEnd.3 HA cells were treated at 1% O 2 concentration for 12 h. Cell viability was detected using the CCK-8 assay. B. Flow cytometry with Annexin V and PI staining was used to determine the apoptotic index of cells. C. WB was used to detect the expression of p53, Bcl-2, Bax, and cleaved caspase3 protein. β-actin was used as an internal control. D. bEnd.3 HA cells were treated at 1% O 2 concentration for 12 h and with LW6, VER-155008, or LW6+VER-155008. Cell viability was detected using the CCK-8 assay. E. bEnd.3 HA cells were treated at 1% O 2 concentration for 12 h and with si- Hif-1α , si- Hspa1a , or si- Hif-1α +si- Hspa1a . Cell viability was detected using the CCK-8 assay. F. Flow cytometry with Annexin V and PI staining was used to determine the apoptotic index of cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Concentration Assay, CCK-8 Assay, Flow Cytometry, Staining, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: Diagram representing the potential mechanism by which HA regulates stabilization of HIF-1α protein via HSP70 in bEnd.3 cells. Under normal conditions, HIF-1α protein is ubiquitinated and degraded by the proteasome. Under HA conditions, HSP70 interacts with the HIF-1α protein, inhibiting its ubiquitination, stabilizing the HIF-1α protein, and promoting the transcription of downstream genes in the nucleus.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Non-cell-autonomous HSC70.1 chaperone displays homeostatic feed-back regulation by binding its own mRNA

doi: 10.1101/2022.05.10.491294

Figure Lengend Snippet: ( A ) Microscale Thermophoresis experiment. In vitro synthesized BAG1 and HSC70 . 1 RNAs ranging between 1 μM and 30 pM were titrated against cell lysates from transgenic plants expressing YFP-HSC70.1 fusion protein. Binding curves were evaluated and plotted as fraction bound against increasing RNA concentrations. Differences in binding are visible as a shift of the sigmoidal binding curve along the x-axis. ( B ) EC50 estimation for both curves. The E C50 of BAG1 towards HSC70.1 is approx. 9 times higher than towards HSC70.1 indicating a higher affinity of HSC70.1 protein towards HSC70 . 1 compared to BAG1 . ( C ) Inhibition of YFP-HSC70 . 1 vs. YFP translation in the presence of increasing concentrations of YFP-HSC70.1 protein. YFP-HSC70.1 was extracted from 10 days old transgenic plants and added to the in vitro wheat germ expression assay translating either YFP (control) or YFP-HSC70.1. Anti-GFP/YFP antibody was used to detect YFP-HSC70.1 protein addded and Green lys was used to detect newly synthesized YFP-HSC70.1 and YFP. ( D ) Relative ratios of in vitro translated YFP-HSC70.1 and YFP protein (see also Fig S6B). ( E ) Model of the predicted effect of HSC70.1 inhibiting its own translation on the refolding of misfolded client proteins. A stress event is assumed to occur (at t=200 s) that gives rise to a sudden increase in misfolded protein. How quickly this amount of misfolded protein deccreases shows how well the HSC70.1 system performs. The model predicts no (blue), slow (red), or a fast (green) refolding of client proteins to due sudden (acute) chaperone demands with no feedback or with translational feedback of HSC70.1 on its own translation (for details see results text, Fig S6C, and Table S3). ( F ) Western Blot (WB) assays on 14 days old YFP, YFP-HSC70.1, and YFP-HSC70.1 ΔSVR transgenic wild-type plants used for HSC70.1 inhibitor (VER-155008) treatment (0, 15 minutes, 30 minutes, and 60 minutes). YFP, YFP-HSC70.1, and YFP-HSC70.1 ΔSVR fusion proteins were detected by YFP antibody. ( G ) Line plot representing average density of bands measured on western blots relative to mock control (n= 3 independent experiments). Significance was calculated using Student’s T-test (two tails); p values indicated by a and b: a,b< 0.001. ( H ) qRT-PCR assays on samples from 14 days old YFP, YFP-HSC70 . 1 , and YFP-HSC70 . 1 ΔSVR transgenic wild-type plants treated with HSC70.1 inhibitor VER-155008 for 0, 15 minutes, 30 minutes, and 60 minutes. Compare with non-treated samples, all treated samples show no significant differences in YFP and and YFP-HSC70 . 1 transcript levels (p values >0.05 ; n = 3 biological replicates; 4 technical replicates). Y axis: relative transcripts levels of YFP and YFP-HSC70 . 1 normalized to UBQ10 . Error bars: Standard Error.Significance was calculated using Student’s T-test (two tails).

Article Snippet: YFP-HSC70.1, YFP-HSC70 ΔSVR, and YFP transgenic plants (14 days after germination on 0.5 MS plates supplemented with 1% sucrose; n>20) were transferred to 5 mL liquid 0.5 MS medium with 1% sucrose and incubated for 15, 30, and 60 minutes with 150 μM HSP70 inhibitor VER-155008 (Sigma SML0271; resuspended in DMSO) or 0.1% DMSO (mock treatment) at 20°C.

Techniques: Microscale Thermophoresis, In Vitro, Synthesized, Transgenic Assay, Expressing, Protein Binding, Binding Assay, Inhibition, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Non-cell-autonomous HSC70.1 chaperone displays homeostatic feed-back regulation by binding its own mRNA

doi: 10.1101/2022.05.10.491294

Figure Lengend Snippet: ( A ) Representative pictures of analyzed wild-type (Col-0), hsc70 . 1, hsp70 . 4, hsc70 . 1 hsp70 . 4, HSC70 . 1 #1 ( hsc70 . 1 hsp70 . 4), HSC70 . 1 #2 ( hsc70 . 1 hsp70 . 4 ), HSC70 . 1M #6 ( hsc70 . 1 hsp70 . 4 ), HSC70 . 1M #9 ( hsc70 . 1 hsp70 . 4 ) and HSC70 . 1M-1 #11 ( hsc70 . 1 hsp70 . 4 ) plants 14 days after germination. ( B ) Quantitative data of measured primary root length of wild-type and indicated mutant plants. Box plot graph: Boxes denote variation between datasets and means; n = number of analyzed plants; error bar: ± SE; black dots: measurements out of ± SE range. Significance was calculated using Student’s T-test (two tails); p value indicated by a and b: a, b<0.001.

Article Snippet: YFP-HSC70.1, YFP-HSC70 ΔSVR, and YFP transgenic plants (14 days after germination on 0.5 MS plates supplemented with 1% sucrose; n>20) were transferred to 5 mL liquid 0.5 MS medium with 1% sucrose and incubated for 15, 30, and 60 minutes with 150 μM HSP70 inhibitor VER-155008 (Sigma SML0271; resuspended in DMSO) or 0.1% DMSO (mock treatment) at 20°C.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Non-cell-autonomous HSC70.1 chaperone displays homeostatic feed-back regulation by binding its own mRNA

doi: 10.1101/2022.05.10.491294

Figure Lengend Snippet: ( A ) Representative pictures of analyzed wild-type (Col-0), hsc70 . 1, hsp70 . 4, hsc70 . 1 hsp70 . 4, HSC70 . 1 #1 ( hsc70 . 1 hsp70 . 4), HSC70 . 1 #2 ( hsc70 . 1 hsp70 . 4 ), HSC70 . 1M #6 ( hsc70 . 1 hsp70 . 4 ), HSC70 . 1M #9 ( hsc70 . 1 hsp70 . 4 ) and HSC70 . 1M-1 #11 ( hsc70 . 1 hsp70 . 4 ) plants 30 days after germination. ( B ) Age of plants at bolding of wild-type and indicated mutant plants. ( C ) Numbers of rosette leaves at time of bolding of wild-type and indicated mutant plants. Box plot graph: Boxes indicate variation between datasets and means; n = number of plants analyzed; error bar: ± SE; black dots: measurements out of range ± SE; significance was calculated using Student’s T-test (two tails); p value indicated by a and b: a, b<0.001.

Article Snippet: YFP-HSC70.1, YFP-HSC70 ΔSVR, and YFP transgenic plants (14 days after germination on 0.5 MS plates supplemented with 1% sucrose; n>20) were transferred to 5 mL liquid 0.5 MS medium with 1% sucrose and incubated for 15, 30, and 60 minutes with 150 μM HSP70 inhibitor VER-155008 (Sigma SML0271; resuspended in DMSO) or 0.1% DMSO (mock treatment) at 20°C.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Non-cell-autonomous HSC70.1 chaperone displays homeostatic feed-back regulation by binding its own mRNA

doi: 10.1101/2022.05.10.491294

Figure Lengend Snippet: ( A ) Representative pictures of root/shoot grafted hsc70 . 1 hsp70 . 4 mutant plants 29 days after grafting. ( B ) RT-PCR assays confirming presence of mobile HSC70 . 1 transcript produced in hsp70 . 4 mutant tissue in heterologous hsc70 . 1 hsp70 . 4 root and shoot tissue (arrowheads). ( C ) Time of flowering (bolding) in days after grafting. Box plots: Boxes indicate the variation between datasets and means; 16 grafted plants were analyzed for each graft combination; error bar: ± SE; black dots: measurements out of range ± SE; significance was calculated using Student’s T-test (two tails); p-value indicated by a and b: a, b<0.001.

Article Snippet: YFP-HSC70.1, YFP-HSC70 ΔSVR, and YFP transgenic plants (14 days after germination on 0.5 MS plates supplemented with 1% sucrose; n>20) were transferred to 5 mL liquid 0.5 MS medium with 1% sucrose and incubated for 15, 30, and 60 minutes with 150 μM HSP70 inhibitor VER-155008 (Sigma SML0271; resuspended in DMSO) or 0.1% DMSO (mock treatment) at 20°C.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Produced