Journal: bioRxiv

Article Title: ER stress and mitochondrial dynamics: a tale of a wandering phosphatase DUSP28

doi: 10.1101/2024.08.17.608394

Figure Lengend Snippet: (A) IFM images of siControl and siDUSP28 transfected cells that were immunostained for mitochondrial protein TOM20. (B) The images of A were quantified to measure the average aspect ratio (arbitrary units, A.U.) and the area of mitochondria per cell, presented as separate graphs. n=3. (C) IFM analysis of control and DUSP28 knockdown cells that were transfected with KDEL-RFP to mark the ER. The images were quantified and measured the percentage area of ER tubules per cell and the fluorescence intensity (CTCF) of KDEL-RFP per cell, plotted and shown in D. n=3. (E) IFM images of control and DUSP28 depleted cells that were stained for ER stress transcription factors, XBP-1, ATF6 and ATF4 to represent the activation of IRE1, ATF6 and PERK pathways, respectively. (F) Graph representing the nuclear localization of ATF4 fluorescence intensity (FI) in the nucleus vs total in siControl and siDUSP28 transfected cells. n=3. (G) Graph representing the quantification of mitochondrial aspect ratio (arbitrary units, A.U.) per cell in control and DUSP28 knockdown cells in the absence and presence of tunicamycin (Tu). n=3. Representative IFM images of control and DUSP28 knockdown cells stained for TOM20 are shown in the bottom most panel of E . All insets of IFM images are amplified white box areas. Scale 10 μm. (H-J) Rescue experiments to demonstrate the specific effect of DUSP28 knockdown on mitochondria using 3’-UTR specific siRNA. The siRNAs were transfected both in HeLa and DUSP28-GFP stable HeLa cells to knock down the endogenous DUSP28. The mRNA levels of DUSP28 were analyzed by agarose gel electrophoresis, and 18S rRNA was used as the internal control (H) . (I) Immunoblotting analysis of DUSP28-GFP levels in control and DUSP28-knockdown (3’-UTR) cells. GAPDH is used as a loading control. *, indicates the non-specific band recognized by anti-GAPDH antibody. (J) Graph representing the quantification of mitochondrial aspect ratio (arbitrary units, A.U.) in each condition described in H / I . n=3. All values in the graphs are presented as mean±s.e.m. *p≤0.05, ***p≤0.001, ****p ≤0.0001, and ns, non-significant.

Article Snippet: The following commercial polyclonal and monoclonal antisera were used in this study: anti-ARF1+ARF3 (Ab32524) and anti-DRP1 (Ab56788) were from Abcam; anti-GM130 (610822) and anti-p230 trans Golgi (611281) were from BD Biosciences; anti-ATF4 (11815), anti-BiP (3177), anti-Calnexin (2679), anti-Phospho-DRP1(S616) (4494), anti-Phospho-DRP1(S637) (6319), anti-EEA1 (3288), anti-IRE1 (3294), anti-MFF (84580), anti-Mitofusin-2 (11925), anti-Parkin (4211) and anti-PERK (5683) were from Cell Signaling Technology; anti-hLAMP-1 (H4A3) was from Developmental Studies Hybridoma Bank; anti-FIS1 (10956-1-AP) was from ProteinTech; anti-ATF-6α (sc-22799), anti-GAPDH (sc-25778), anti-Tom20 (sc-17764) and anti-XBP-1 (sc-7160) were from Santa Cruz Biotechnology; anti-β-Actin (A5441), anti-LC3B (L7543), and anti-γ-Tubulin (T6557, IB:1:10000) from Sigma-Aldrich (Merck); and anti-GFP (A-11122) was from Thermo Fisher Scientific (Invitrogen).

Techniques: Transfection, Control, Knockdown, Fluorescence, Staining, Activation Assay, Amplification, Agarose Gel Electrophoresis, Western Blot