Journal: bioRxiv

Article Title: Lysosomal MLKL is balanced by ESCRT to control cell death

doi: 10.1101/2023.08.29.555049

Figure Lengend Snippet: ( a ) Western blot analysis of SH-SY5Y cells primed for 16 h with IFN type I followed or not by treatment with Poly(I:C) (IFN/pIC) for 48 h using anti-MLKL, -RIPK3 and -actin antibodies. ( b and c ) MLKL was knocked out by CRISPR/Cas9 approach using two different gRNAs in SH-SY5Y cells. ( b ) Western blot analysis of MLKL expression levels using anti-MLKL and -actin antibodies treated with IFN for 16 h. ( c ) Cell death profile following IFN/pIC treatment for 48 h analyzing SytoxGreen (SG) positive cells. Data points represent the mean ± □S.E.M. of n = 3. **p = 0.009 and 0.0032. ( d ) Cell death profile analyzing PI positive SH-SY5Y cells, left untreated or treated for 48 h with IFN/pIC in combination with GSK-872’ (RIPK3i) or necrosulfonamide (NSA). Data points represent the mean ± S.E.M. of four independent experiments (n = 4); ***p=0.0004; ns= not significant. ( e ) Phostag SDS-Page of SH-SY5Y cells treated for indicated times with IFN/pIC or HT-29 cells with pBZ for 12 h to induce necroptosis using anti-p-MLKL (T357/S358) or anti-MLKL antibody. Phospho-MLKL bands were verified by lambda phosphatase treatment. A representative of n = 3 is shown. ( f ) Western blot analysis under non-reducing (-DTT) and reducing (+DTT) conditions of SH-SY5Y cells treated with IFN/pIC for the indicated times using an anti-MLKL antibody. A representative of n=3 is shown; left panel. Oligomeric MLKL is indicated. Right panel: Western blot analysis under non-reducing conditions of HT-29 cells treated with pBZ (poly(I:C), BV6 and zVAD-fmk; necroptosis (nec)) for 12 h using an anti-MLKL antibody. Oligomeric MLKL is indicated by an arrowhead. b: bottom of the well; i: interface between stacking and running gel. ( g ) Confocal images of MLKL-GFP expressing SH-SY5Y cells expressing LAMP-1-RFP and Hoechst left untreated or treated for 24 h with IFN/pIC. Merged image of MLKL-GFP, LAMP-1 and Hoechst right panel. Scale bars 10 µm. Representative images of n = 1 are shown. ( h ) Quantification of ( g ) analyzing the percentage of overlay of LAMP-1-RFP fluorescence intensity with MLKL-GFP. Data points represent the mean ± S.E.M. of n = 2 analyzing at least 25 cells. ****p < 0.0001.

Article Snippet: For lambda phosphatase treatment cells were lysed in RIPA buffer and incubated with lambda phosphatase (New England biolabs; P0753S) for 30 min at 37 °C prior to TCA precipitation and loading on Phostag SDS-Page.

Techniques: Western Blot, CRISPR, Expressing, SDS Page, Fluorescence

Journal: Biosensors

Article Title: Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe

doi: 10.3390/bios12080569

Figure Lengend Snippet: Feasibility of M.SssI detection with RCA. ( A ) Fluorescence emission spectra with or without M.SssI following the addition of SYBR TM Gold. ( B ) Polyacrylamide gel electrophoresis supports the feasibility of the strategy: (Lane M) 20 bp DNA marker; (Lane a) PPs; (Lane b) PPc; (Lane c) PPs + PPc + T4 DNA ligase; (Lane d) PPs + PPc + T4 DNA ligase + HpaII + lambda exo + ExoI; (Lane e) PPs + PPc + T4 DNA ligase + M.SssI + HpaII + lambda exo+ ExoI.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/bios12080569/s1 , Table S1: Oligonucleotides used in this study; Table S2: Detection limits and real sample applications of some MTase detection methods; Figure S1: Electrophoresis analysis of the products of RCA of the detection system with (Lane a) and without (Lane b) M.SssI by RCA (Lane M) 15,000 bp DNA marker; Figure S2: Optimize the methylation and cleavage buffers. (A) F is the fluorescence intensity with M.SssI and F 0 is fluorescence intensity without M.SssI. (B) The fluorescence intensities of the sensing systems with or without M.SssI. (a) CutSmart buffer (methylation process) + CutSmart buffer (cleavage process); (b) NEBuffer 2 (methylation process) + CutSmart buffer (cleavage process); (c) NEBuffer 2(methylation process) + CutSmart buffer + lambda reaction buffer (cleavage process); (d) CutSmart buffer (methylation process) + lambda reaction buffer (cleavage process); Figure S3: Optimize the time of methylation process; Figure S4: Optimize the time of cleavage of HpaII and lambda exo; Figure S5: Optimize the time of digestion of ExoI; Figure S6: Eliminate the probable impact of detection system.

Techniques: Fluorescence, Polyacrylamide Gel Electrophoresis, Marker