John Blair
@jblairsci.bsky.social
Postdoc in the Satija Lab at NYGC. Former PhD at Bateup Lab at UC Berkeley
This paper was entirely led by Izzy and I was an accessory in the wet lab performing the necessary validation experiments alongside others - its exciting stuff!
October 23, 2025 at 4:42 PM
This paper was entirely led by Izzy and I was an accessory in the wet lab performing the necessary validation experiments alongside others - its exciting stuff!
She observed that the knocking down RPL10 and RPL24 has the same effect as knocking down RPL5 and RPL11, known members of the ribosomal stress pathway, showing that her fingerprinting-identified candidates were indeed real!
October 23, 2025 at 4:42 PM
She observed that the knocking down RPL10 and RPL24 has the same effect as knocking down RPL5 and RPL11, known members of the ribosomal stress pathway, showing that her fingerprinting-identified candidates were indeed real!
Next, she identified two proteins that were likely involved in the ribosomal stress response from their transcriptome profile. We used FlexPlex to knock down those genes with siRNA, treated them with Nutlin-3a, oligohashed them and then used custom probes to confirm the loss of the intended target.
October 23, 2025 at 4:42 PM
Next, she identified two proteins that were likely involved in the ribosomal stress response from their transcriptome profile. We used FlexPlex to knock down those genes with siRNA, treated them with Nutlin-3a, oligohashed them and then used custom probes to confirm the loss of the intended target.
From there she determined that only under certain doses were these inhibitors HDAC-specific and above a certain threshold, they then act like pan-HDAC inhibitors
October 23, 2025 at 4:42 PM
From there she determined that only under certain doses were these inhibitors HDAC-specific and above a certain threshold, they then act like pan-HDAC inhibitors
First, she was curious about the specificity of HDAC inhibitors that are purported to be HDAC subclass specific versus pan-HDAC inhibitors, so we applied FlexPlex with oligo-based hashing to simultaneously observe the effect of these compounds on the transcriptome and histone modifications
October 23, 2025 at 4:42 PM
First, she was curious about the specificity of HDAC inhibitors that are purported to be HDAC subclass specific versus pan-HDAC inhibitors, so we applied FlexPlex with oligo-based hashing to simultaneously observe the effect of these compounds on the transcriptome and histone modifications
There is a lot in this paper and I won't go into the method, but will highlight how we applied FlexPlex within the manuscript to help validate some of the phenomena that Izzy discovered using RNA fingerprinting
October 23, 2025 at 4:42 PM
There is a lot in this paper and I won't go into the method, but will highlight how we applied FlexPlex within the manuscript to help validate some of the phenomena that Izzy discovered using RNA fingerprinting
Overall, this paper was led by myself and carried out by a number of people including @abradu.bsky.social , Izzy Grabski, Carol Dalgarno and of course Rahul Satija.
October 23, 2025 at 4:26 PM
Overall, this paper was led by myself and carried out by a number of people including @abradu.bsky.social , Izzy Grabski, Carol Dalgarno and of course Rahul Satija.
We also identified new candidates for involvement in the integrated stress response, including RACK1, showing an increase in ATF4 levels upon perturbation
October 23, 2025 at 4:26 PM
We also identified new candidates for involvement in the integrated stress response, including RACK1, showing an increase in ATF4 levels upon perturbation
One observation of interest was a set of genes that seemingly responded to feedback from mTOR signaling - going up with both Ragulator and TSC loss and going down with mTORC1 and GATOR1 loss - sets of complexes that have opposite effects on mTOR signaling
October 23, 2025 at 4:26 PM
One observation of interest was a set of genes that seemingly responded to feedback from mTOR signaling - going up with both Ragulator and TSC loss and going down with mTORC1 and GATOR1 loss - sets of complexes that have opposite effects on mTOR signaling
Finally, we clustered perturbations from the validation FlexPlex assay into different categories based on their transcriptome and we observed how the different perturbations converge on mTOR signaling
October 23, 2025 at 4:26 PM
Finally, we clustered perturbations from the validation FlexPlex assay into different categories based on their transcriptome and we observed how the different perturbations converge on mTOR signaling
We validated 120 of these hits with another FlexPlex assay, observing pRPS6 changes in most of them.
October 23, 2025 at 4:26 PM
We validated 120 of these hits with another FlexPlex assay, observing pRPS6 changes in most of them.
However, we wanted to bring these predictions genome-wide - so we applied the model to the K562 GWPS dataset from the Weissman Lab, observing 586 perturbations in that dataset that we predicted to significantly alter pRPS6
October 23, 2025 at 4:26 PM
However, we wanted to bring these predictions genome-wide - so we applied the model to the K562 GWPS dataset from the Weissman Lab, observing 586 perturbations in that dataset that we predicted to significantly alter pRPS6
From there we modeled pRPS6 activity based on transcriptomic profile using RIDGE regression and applied it to other datasets from with known or inferred changes in pRPS6 - including datasets in completely different cell types including organoids!
October 23, 2025 at 4:26 PM
From there we modeled pRPS6 activity based on transcriptomic profile using RIDGE regression and applied it to other datasets from with known or inferred changes in pRPS6 - including datasets in completely different cell types including organoids!
We then curated that list to 50 of the top perturbations producing higher or lower pRPS6 and ran a FlexPlex assay with that, producing a “glossary” dataset with paired transcriptome-pRPS6 profiles for each perturbation.
October 23, 2025 at 4:26 PM
We then curated that list to 50 of the top perturbations producing higher or lower pRPS6 and ran a FlexPlex assay with that, producing a “glossary” dataset with paired transcriptome-pRPS6 profiles for each perturbation.
We then applied FlexPlex to understand regulators of mTORC1 signaling. We first did a genome-wide bulk CRISPRi screen on pRPS6 in K562 cells to provide a candidate list of relevant perturbations which, while noisy, reconstructed the known mTOR pathway and revealed new potential regulators.
October 23, 2025 at 4:26 PM
We then applied FlexPlex to understand regulators of mTORC1 signaling. We first did a genome-wide bulk CRISPRi screen on pRPS6 in K562 cells to provide a candidate list of relevant perturbations which, while noisy, reconstructed the known mTOR pathway and revealed new potential regulators.
We demonstrate the effectiveness of FlexPlex through perturbing known components of the mTOR pathway, observing increases in pRPS6 in cells with TSC1 knocked down and decreases in cells with RPTOR knocked down. We confirmed excellent guide assignment and robust transcriptome profiling with high UMIs
October 23, 2025 at 4:26 PM
We demonstrate the effectiveness of FlexPlex through perturbing known components of the mTOR pathway, observing increases in pRPS6 in cells with TSC1 knocked down and decreases in cells with RPTOR knocked down. We confirmed excellent guide assignment and robust transcriptome profiling with high UMIs
March 9, 2025 at 6:16 AM
I will make Helen and Tommy get bluesky and then i will @ them in a subsequent post, but thank you for reading!!
March 6, 2025 at 9:42 PM
I will make Helen and Tommy get bluesky and then i will @ them in a subsequent post, but thank you for reading!!
This work was the last year or so of my Ph.D in the Bateup Lab as covid hit, and I didn’t have an opportunity to complete it.
Very fortunately, Tommy came along and added SO much (immunopanning, 4i, additional scRNA-seq, westerns and all the analysis) making it way better than i ever could have!
Very fortunately, Tommy came along and added SO much (immunopanning, 4i, additional scRNA-seq, westerns and all the analysis) making it way better than i ever could have!
March 6, 2025 at 9:42 PM
This work was the last year or so of my Ph.D in the Bateup Lab as covid hit, and I didn’t have an opportunity to complete it.
Very fortunately, Tommy came along and added SO much (immunopanning, 4i, additional scRNA-seq, westerns and all the analysis) making it way better than i ever could have!
Very fortunately, Tommy came along and added SO much (immunopanning, 4i, additional scRNA-seq, westerns and all the analysis) making it way better than i ever could have!
There is a ton of IF showing the remarkable pathology of both the knockout organoid astrocytes (left) and astrocytes from resected patient cortical tuber tissue
March 6, 2025 at 9:42 PM
There is a ton of IF showing the remarkable pathology of both the knockout organoid astrocytes (left) and astrocytes from resected patient cortical tuber tissue
The single cell sequencing was performed at multiple important timepoints in early development and in multiple genetic backgrounds, all with our meticulously engineered conditional two-hit TSC2 knockout alleles and knockout reporter cassettes
March 6, 2025 at 9:42 PM
The single cell sequencing was performed at multiple important timepoints in early development and in multiple genetic backgrounds, all with our meticulously engineered conditional two-hit TSC2 knockout alleles and knockout reporter cassettes
Also I couldn't have done it without the fellowship support of the @jcchildsfund.bsky.social - thank you!
February 28, 2025 at 4:41 PM
Also I couldn't have done it without the fellowship support of the @jcchildsfund.bsky.social - thank you!