Fig 2: Manipulating the expression of Zbtb7a in WT and TG2-/- astrocytes differentially impacts morphology. a. Immunoblots showing knockdown (KD) or overexpression (OE) of Zbtb7a, compared to control (CTL), in WT and TG2-/- astrocytes. Since the Zbtb7a OE virus encodes human Zbtb7a, the Zbtb7a bands in these samples migrated slightly less far down the gel than bands in the CTL and KD samples. b. Representative images of transduced astrocyte GFAP network (scale bar = 20 µm) and c. Quantitation of GFAP network area (n=34–50 astrocytes per condition, **p<0.0001), ****p<0.0001).

Fig 3: TG2 and Zbtb7a interact. a. Nuclear (Nuc) and cytosolic (Cyto) fractions of wild type (WT) and TG2 knockout (TG2-/-) astrocytes. Intensity of bands are not representative of the proportion of the total protein in the cytosolic/nuclear fractions. b. ICC showing co-localization of TG2 and Zbtb7a in nucleus of astrocytes. Quantification of co-localization of nuclear TG2 and Zbtb7a signal with the median Manders’ coefficient and 95% CI plotted (n=30). c. Input controls of V5-TG2 and FLAG-Zbtb7a (FL) transfected in HEK293TN cells. Immunoprecipitation of V5-TG2 pulls down FLAG-Zbtb7a. d. Immunoprecipitation of endogenous TG2 from astrocyte nuclear fraction pulls down Zbtb7a. Probes for TG2 and Zbtb7a from the same blot shown with different exposures.

Fig 4: TG2-/- astrocytes promote neurite outgrowth to a greater extent than WT astrocytes on an inhibitory matrix, a. Representative MAP2 images of neurons grown on PDL (-) or PDL+CSPG (+) matrix and paired with WT or TG2-/- astrocytes (scale bar = 20 µm) b. Quantitation of neurite length on CSPG (n=39–57 neurons per condition, *p<0.05, **p<0.01, ***p<0.001). c. Quantitation of primary neurite number on CSPG (n=43–68 neurons per condition, ***p<0.001).

Fig 5: Astrocytes facilitate synapse formation among neurons on a permissive, but not inhibitory matrix, independent of the presence of TG2. a. Images of PSD95/Synaptophysin immunostaining of neurons grown on PDL without astrocytes or with WT or TG2-/- astrocytes (scale bar = 20 µm), b. Images of PSD95/synaptophysin immunostaining of neurons grown on CSPG inhibitory matrix without astrocytes or with WT or TG2-/- astrocytes (scale bar = 20 µm), c. Quantification of neuronal synapses approximated by PSD95 puncta colocated with Synaptophysin puncta exclusively in neurites (18–24 neurons per group, ****p<0.001). d. Representative images of Homer/Bassoon immunostaining for neurons grown on PDL and PDL+CSPG (scale bar = 20 µm), and quantification of synapses on PDL approximated by Homer puncta colocated with Bassoon puncta exclusively in neurites (22–23 neurons per group, ***p<0.001).