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. 2023 Jul 5;42(16):e112812. doi: 10.15252/embj.2022112812

Figure EV4. Endosome displacement and Golgi dispersion do not rely on tubulin acetylation.

Figure EV4

  1. Quantification of αTAT1 mRNA expression in cells treated with siRNA against αTAT1 (two independent siRNAs; #5 and #9).
  2. Left panel; immunoblot for α‐tubulin and acetylated tubulin (Ac‐tub) upon αTAT1 depletion (two independent siRNAs; #5 and #9). Right panel; percentage of acetylated tubulin relative to total α‐tubulin.
  3. Representative images of cells stained for microtubules (α‐tubulin; gray), acetylated tubulin (Ac‐tubulin, orange) and DNA (Hoechst, cyan) upon αTAT1 depletion. Scale bar: 20 μm.
  4. Representative images of cells stained for early endosomes (EEA1, orange), F‐actin (phalloidin, gray), and DNA (Hoechst, cyan) upon αTAT1 depletion. Scale bar: 10 μm.
  5. Quantification of endosome‐nucleus distance (Left panel: n (−DOX siCtr) = 88; n (+DOX siCtr) = 90; n (−DOX siαTAT1#5) = 91; (+DOX siαTAT1#5) = 98; Right panel: n (−DOX siCtr) = 70; n (+DOX siCtr) = 64; n (−DOX siαTAT1#9) = 69; (+DOX siαTAT1#9) = 67).
  6. Representative images of cells stained for Golgi (GM130, orange), F‐actin (phalloidin, gray), and DNA (Hoechst, cyan) upon αTAT1 depletion. Scale bar: 10 μm.
  7. Quantification of Golgi area (Left panel: n (−DOX siCtr) = 177; n (+DOX siCtr) = 147; n (−DOX siαTAT1#5) = 172; n (+DOX siαTAT1#5) = 156; Right panel: n (−DOX siCtr) = 103; n (+DOX siCtr) = 103; n (−DOX siαTAT1#9) = 128; n (+DOX siαTAT1#9) = 109).
  8. Representative images of cells treated with siRNA against αTAT1, stained for microtubules (α‐tubulin, cyan), acetylated tubulin (Ac‐tubulin, orange) and DNA (Hoechst, gray) upon nocodazole treatment (Noc, 2 μM). Scale bar: 10 μm.
  9. Quantification of microtubule numbers (n (+DOX siCtr+Noc) = 145; n (+DOX siαTAT1+Noc) = 159).

Data information: For all graphs, error bars represent mean ± SD from three independent experiments. P‐values are described in the graphs, ns = not significant (P > 0.05). The following statistics were applied: one‐way ANOVA with Tukey's post hoc test for graphs in (E) and (G) and unpaired t‐test for graph in (I). n = number of cells analyzed.

Source data are available online for this figure.